US 2010.0075894A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0075894 A1 Hotamisligil et al. (43) Pub. Date: Mar. 25, 2010

(54) REDUCINGER STRESS IN THE TREATMENT A63L/92 (2006.01) OF OBESITY AND DABETES A 6LX 3/575 (2006.01) A63/04 (2006.01) (75) Inventors: Gökhan S. Hotamisligil, Wellesley, A6II 3/55 (2006.01) MA (US); Umut Ozcan, Brookline, A613/60 (2006.01) MA (US) A6IP3/10 (2006.01) A6IP3/04 (2006.01) Correspondence Address: A6IP 9/10 (2006.01) EDWARDSANGELL PALMER & DODGE LLP (s2 usic... 514/4; 435/7.2:436/86; 514/570; P.O. BOX SS874 514/169; 514/740: 514/171; 514/635: 514/165 BOSTON, MA 02205 (US) (57) ABSTRACT (73) Assignee: HARYARPN.YERSITY, Endoplasmic reticulum stress has been found to be associated ambridge, (US) with obesity. Therefore, agents that reduce or prevent ER (21) Appl. No.: 12/541,020 stress may be used to treat diseases associated with obesity including peripheral insulin resistance, hypergylcemia, and (22) Filed: Aug. 13, 2009 type 2 diabetes. Two compounds which have been shown to reduce ER stress and to reduce blood glucose levels include Related U.S. Application Data 4-phenyl butyric acid (PBA), tauroursodeoxycholic acid (TUDCA), and trimethylamine N-oxide (TMAO). Other (63) Continuation of application No. 1 1/227.497, filed on compounds useful in reducing ER stress are chemical chap Sep. 15, 2005, now abandoned. erones Such as trimethylamine N-oxide and glycerol. The (60) Provisional application No. 60/610,093, filed on Sep. E. invention provides methods of treating a subject suf 15, 2004. ering from obesity, hyperglycemia, type 2 diabetes, or insu s lin resistance using ER stress reducers such as PBA, TUDCA, O O and TMAO. Methods of screening for ER stress reducers by Publication Classification identifying agents that reduce levels of ER stress markers in (51) Int. Cl. ER stressed cells are also provided. These agents may find use A6 IK 38/28 (2006.01) in methods and pharmaceutical compositions for treating GOIN 33/53 (2006.01) obesity-associated diseases.

LEAN Ob/Ob RD HFD

3. sik--XBP-1(S) GRP78

28S 18S

LEAN Ob/Ob IGRP78

28S --Tot-JNK 18S Patent Application Publication Mar. 25, 2010 Sheet 1 of 22 US 2010/0075894 A1

qO/GO

qO/qONWET

Patent Application Publication Mar. 25, 2010 Sheet 2 of 22 US 2010/0075894 A1

IP: IRS-1 E. E.s R.ss IB:IP: IRS-1IRS-1 ser307 IB: IRS-1 ser307 AIP: IRS-1 IP: IRS-1 EgySESSIB: IRS-1 EIB. IRS-1

IP: pY IB: IRS-1 IP: pY IB: pY Patent Application Publication Mar. 25, 2010 Sheet 3 of 22 US 2010/0075894 A1

is is

2,: ESSEEE i E.SR g Tot-JNK O 1 h FIG. 3A

Tun: IP: IRS-1 s IB: p-IRS-1Ser307 IP: IRS-1 *." IB: IRS-1

IB: p-IRS-1 ser307

IP: IRS-1 IB: IRS-1 Patent Application Publication Mar. 25, 2010 Sheet 4 of 22 US 2010/0075894 A1

IRE-1Ot/ IRE-1O/ Ins: ------

IP: IRS-1 IB: pY IP: IRS B: IRS-1

N - DIRE-1 +/- 5g.DN OIRE-1 -/- O.8 25U. 0.4 O.O Patent Application Publication Mar. 25, 2010 Sheet 5 of 22 US 2010/0075894 A1

3OO U) g 250 c 3 S. S 2 15O O Ye - O 1OO nS 5O

O X PBS 4-PBA FIG. 4A

350 300 g a 250 -

3O N.G 200

t5 a1E 150 g 1OO O

O 15 3O 6O 90 12O Minutes After Insulin Injection FIG. 4B Patent Application Publication Mar. 25, 2010 Sheet 6 of 22 US 2010/0075894 A1

Body Weight During Trial

ex ------V ------a ma ------mava --- m V-w 500 OO S. 50.00 ... -- ob/ob PBS si cleanPBS Eg 40.00-TV. to -A--- ob/oblean PBS PBA 3350- 300 His ean PBA g 30.00 * -e- lean PBA S 58 & 20.00 ------r 3 ra 15 & 10.00 as 19 O.OO 8 R O Day 0 Day 20 S S R Days A FIG. 5B so. ITT at 15th day. Insulin Levels i 300 14 s -s 12 ob/ob PBS is 250 510 Zob/ob PBA 200- -- ob/ob pbs S. Olean PBS Ne -- ob/ob pba S 8 Sean PBA S; 150 -A-lean pbs 6 8 100 r-e-lean pba 4 5 50 - 2 8 O O i O 30 60 90 120 Minutes After Insulin Injection FIG. 5D Patent Application Publication Mar. 25, 2010 Sheet 7 of 22 US 2010/0075894 A1

Blood Glucose Levels

ob/ob(PBS) 4 O O ob/ob(TUD) O Lean (PBS) Lean (TUD) 2 O O

54O ckk ck k ck >k 450 KK ck k ckk -- ob/ob (TUDCA) 36O -H ob/ob (PBS) -A- Lean (PBS) 27O -X - Lean (TUDCA) 18O

9 O

O O 15 3O 60 90 120 Minutes After Insulin Injection FIG. 6B Patent Application Publication Mar. 25, 2010 Sheet 8 of 22 US 2010/0075894 A1

5OO

400

3OO

2OO

1OO

Days of treatment of diabetic mice with TMAO FIG. 7A

16. O PBS OTMAO S. 12.. O - s 8. O

O39 Na

Days of treatment of diabetic mice with TMAO FIG. 7B Patent Application Publication Mar. 25, 2010 Sheet 9 of 22 US 2010/0075894 A1

6 O O

3O O 200 1 OO

O PBS PBA (200mg) MET (200mg) PBA--MET (200) Treatment (diabetic mice, ob/ob, treated for 11 days at 200 mg/kg/day dose)

Glu (mM) STZ. al --

O 5 10 25 75

var-Y mall GRP78 sausales as 28s is 18S Patent Application Publication Mar. 25, 2010 Sheet 10 of 22 US 2010/0075894 A1

FIG. 1 OA

Lean Ob/Ob

Patent Application Publication Mar. 25, 2010 Sheet 11 of 22 US 2010/0075894 A1

IB: pY

: IB: IRS-1Pser a , IP: IRS-1 s IB : IRS-1

Control Insulin s O Ins+Tun O 1OO 200 Phospho/Total

?yS 200 k IP: IRS-1 IB: IRS-1Pser 'g. 120 s IP: IRS-1 th 40 IB: IRS-1 Thap: - - + + JNKi: - - - -

IP: pY IB: IRS-1 .. IP: pY IB: pY Patent Application Publication Mar. 25, 2010 Sheet 12 of 22 US 2010/0075894 A1

TUn: ------Time(hr): O 1. 2 4 p-c-Jun

- IP: IRS-1 : IB: IRS-1 pSer307 IP: IRS-1 IB: IRS-1

p-c-Jun s IP: IRS-1 IB: IRS-1 pSer307 IP: IRS-1 IB: IRS-1

IRE-1Ot/ IRE-O/ Tun; O O 1 2 3 4 O O 1 2 3 4 (hr) Ins: - - - - IP: IRS-1 IB; pY

IP: IRS-1 IB: IRS-1

5OO IRE1 -/- k

4OO IRE-1 - A.A -- 3OO i 2OO 1OO

Tun: O O 1. 2 3 4 (hr) Ins: - H ------FIG. 11H Patent Application Publication Mar. 25, 2010 Sheet 13 of 22 US 2010/0075894 A1

: IRS-1 : pY : IRS-1 : IRS-1 pSer307 : IRS-1 : IRS-1

IR : pY : IR : IR

IP: IRS-1 IB: IRS-1 pSer307

IP: IRS-1 IB : IRS-1

FIG. 12B Patent Application Publication Mar. 25, 2010 Sheet 14 of 22 US 2010/0075894 A1

- FDOX- -DOX Tun(2g/ml): - + + + + + - + + + + + Time(hr): O 1 2 3 4 5 O 1 2 3 4 5 FIG. 13A

--DOX -DOx Tun (2ug/ml): - + + + + + + - + + + + + + Time(hr): O 0.51 2 3 4 5 O 0.51 2 3 4 5

: - - JNK FIG. 13C

XBP-1/ XBP-t/- Tun(O.5ug/ml): - + + + + + + - + + + + + + Time(hr): O O.5 1 2 3 4 5 O 0.51 2 3 4 5

FIG. 13D Patent Application Publication Mar. 25, 2010 Sheet 15 of 22 US 2010/0075894 A1

--DOX -DOX Tun (2g/ml): - + + - - - -- Time(hr): O 4 5 O 4 5 S 1000 D-Dox Y pSer307 '3. 600 ", 400 U?) St IRS-1 a 200 FIG 13E Time(hr): O +Dox 500 400 Uh 300 9, 200 1OO i Time(hr): 5 Tun: - -H Ins: - H

XBP-1-/- XBP-1t/t EXBP-1 -/- N Tun (O.5ug/ml): - + + - + + O 2500 XBP-1 /+ Time(hr): O 4 5 O 4 5 8 2000 w IRS-1 S. 15OO H OOO U : g 5OO

Time(hr): O 4 5 Tun: - -- -- Ins: - -- -- FIG. 13H Patent Application Publication Mar. 25, 2010 Sheet 16 of 22 US 2010/0075894 A1

--Dox -DOX Tun (2g/ml): O O 0.5 1 2 3 4 O O 0.5 1 2 3 4 (hr) InS: ------Ogg Qup

XBP-1/- XBP-1+/+ Tun (0.5ug/ml): O O O.5. 1 2 3 4 O O 0.5 1 2 3 4 (hr) Ins: ------s AAas? as, ? IP:IB: IRpy

IP: IR IB: IR

FIG. 14B Patent Application Publication Mar. 25, 2010 Sheet 17 of 22 US 2010/0075894 A1

3 sks: 18O s

kick S 2 kk 1. 2 O S. k 1 . 6 O O 6 O O O O O 4 8 1216 20 O 4 8. 21620 O 4 8 121620 O 4 8 12 1620 Weeks on Diet Weeks on Diet Weeks on Diet Weeks on Diet FIG. 15A FIG. 15C FIG. 15D 540 540 240 240 .." ck/X sk sk kk 5 3 6 O Yag360 k 1. 6 O sk o 8 O 8 18O 8 O 8 O P (

O O O O O 1530. 6090120 O 1530 6090120 O 1530 60 90120 O 1530 60 90120 Time After Glucose (min) Time After Glucose (min) Time After Insulin (min) Time After Insulin (min) FIG. 15E FIG. 15F FIG. 15G FIG. 15H Patent Application Publication Mar. 25, 2010 Sheet 18 of 22 US 2010/0075894 A1

2100- XBP-1 +/-- 2. 5 XBP-1 +/- 2 XBP-1 +/- O XBP-1 +/- 2 g 1400 D c) 1 5 700 Ol 3. 1 U O

FIG. 1.6A FIG. 16B

140 22 1.8 OO 1. O 5 1. 4. O 7 O

7 O -H XBP-1 +/- 3 5 -o- XBP-1 +/- O O O 15 3O 6O 90 120 1 2 3 4 5 6 Time After Insulin (min) Time After Insulin (min)

FIG. 16C FIG. 16D Patent Application Publication Mar. 25, 2010 Sheet 19 of 22 US 2010/0075894 A1

GS3 W. SEAN ÉS: R FIG. 17

XBP-1+/- XBP1 Eisge:S35

FIG. 17D FIG. 17E

1.8

S 1.2 S 0.6 iii O O 2 5 10 15 20 30 60 HFD Time After Glucose (min) FIG. 17I Patent Application Publication Mar. 25, 2010 Sheet 20 of 22 US 2010/0075894 A1

XBP-1/

FIG. 18A FIG. 18B

XBP-1/- XBP-1/ XBP-1/- Ins: -- -H IP: IRS-1 IB: IRS-1 pSer307 IP: IRS-1 IB: IRS-1 FIG. 18C FIG. 18D

XBP-1/ XBP-1 +/- Ins: - - - InS: - H ------IP: IRS-1 IP: IRS-2 IB: pY IB: pY

as sea is in a varia IP: IRS-1 IP: IRS-2 IB: IRS-1 IB: IRS-2 FIG. 18E FIG 18F XBP-1+/- Ins: ------AKT pSer473

Patent Application Publication Mar. 25, 2010 Sheet 21 of 22 US 2010/0075894 A1

Liver FAT

XBP-1+/* XBP-1+/- XBP-1/+ XBP-1+/- Ins: ------Ins: ------: IR : IR : pY : pY : IR : IR IR : IR

: IRS-1 : IRS-1 : pY : pY : IRS-1 : IRS-1 : IRS-1 : IRS-1

: IRS-2 : IRS-2 : pY : pY

: IRS-2 : IRS-2 : IRS-2 : IRS-2

AKTpser473 AKTpser473

: AKT : AKT s 3 s . . FIG. 19A FIG. 19B Patent Application Publication Mar. 25, 2010 Sheet 22 of 22 US 2010/0075894 A1

XBP-1+/+ XBP-1/ Ins: ------

AKTpSer473

IB: AKT FIG. 2 OA

XBP-1 / XEP-1t/

FIG. 2 OB

wt (pbs) wt (pba) ob/ob (pbs) ob/ob (pba)

P-C-UN FIG. 21. US 2010/0075894 A1 Mar. 25, 2010

REDUCINGER STRESS IN THE TREATMENT which is incorporated herein by reference). To cope with ER OF OBESITY AND DIABETES stress, cells activate a signal transduction system linking the ER lumen with the cytoplasm and nucleus, called the RELATED APPLICATIONS unfolded protein response (UPR) (Hampton Curr. Biol. 0001. The present application claims priority under 35 10:R518, 2000; Mori Cell 101:451, 2000; Harding et al. U.S.C. S 119(e) to U.S. provisional patent application, U.S. Annu. Rev. Cell Dev. Biol. 18:575, 2002; each of which is Ser. No. 60/610,093, filed Sep. 15, 2004, which is incorpo incorporated herein by reference). Among the conditions that rated herein by reference. This application is also related to trigger ER stress are glucose and nutrient deprivation, Viral U.S. provisional patent application, U.S. Ser. No. 60/610, infections, increased synthesis of secretory proteins, and the 286, filed Sep. 15, 2004, entitled “Modulation of XBP-1 expression of mutant or misfolded proteins (Ma et al. Cell activity for treatment of metabolic disorders'; U.S. patent 107:827, 2001; Kaufman et al. Nat. Rev. Mol. Cell. Biol. application, U.S. Ser. No. filed Sep. 15, 2005, 3:411, 2002; each of which is incorporated herein by refer entitled “Modulation of XBP-1 activity for treatment of meta ence). bolic disorders'; and U.S. patent application, U.S. Ser. No. 10/655,620, filed Sep. 2, 2003, entitled “Methods and Com positions for Modulating XBP-1 Activity”, each of which is SUMMARY OF THE INVENTION incorporated herein by reference. 0006. Many of the conditions that have been shown to GOVERNMENT SUPPORT trigger ER stress have also been found to occur in obesity, and associated diseases such as type 2 diabetes, hyperglycemia, 0002. The work described herein was supported, in part, and insulin resistance. For example, obesity increases the by a grant no. 32412 from the National Institutes of Health. demand on the synthetic machinery of the cell in many secre The United States government may have certain rights in the tory organ systems and is also associated with abnormalities invention. in intracellular energy fluxes and nutrient availability. The present invention stems from the recognition that many of BACKGROUND OF THE INVENTION these diseases associated with obesity cause ER stress, par 0003. The dramatic increase in the incidence of obesity in ticularly in peripheral tissues, and that ER stress is involved in most parts of the world has contributed to an increased inci triggering insulin resistance and type 2 diabetes, two sequelae dence of insulin resistance, type 2 diabetes, and cardiovascu of obesity. Therefore, agents that reduce ER stress are useful lar disease. These obesity-associated diseases have become in treating obesity, peripheral insulin resistance, hyperglyce serious threats to human health. mia, and type 2 diabetes. The agents useful in the treatment of 0004 Obesity has been found to be associated with the these diseases include small molecules, proteins, nucleic activation of cellular stress signaling pathways (Uysal et al. acids, and any other chemical compounds known to reduce or Nature 389:610, 1997: Hirosumi et al. Nature 420:333, 2003: prevent ER stress. These agents make act in any manner that Yuan et al. Science 293:1673, 2001; each of which is incor reduces or prevents ER stress such as reducing the production porated herein by reference). One player in the cellular stress of mutant or misfolded proteins, increasing the expression of response is the endoplasmic reticulum (ER), a membranous ER chaperones, increasing the stability of proteins, boosting network that functions in the synthesis and processing of the processing capacity of the ER, etc. Particularly useful secretory and membrane proteins. The ER is responsible for agents include chemical chaperones such as 4-phenyl the processing and translocation of most secreted and integral butyrate (PBA), tauroursodeoxycholic acid (TUDCA), trim membrane proteins of eukaryotic cells. The lumen of the ER ethylamine N-oxide (TMAO), glycerol, DO, dimethylsu provides a specialized environment for the posttranslational floxide, glycine betaine, methyl amines, and glycerophos modification and folding of these proteins. Properly folded phocholine. In particular, both PBA and TUDCA have been proteins are cleared for exit from the ER and progress down shown to regulate ER stress in animals as measured by the the secretory pathway, while unfolded or misfolded proteins reduced phosphorylation of PERK, reduced activation of are disposed of by ER-associated protein degradation JNK, and reduced phosphorylation of IRE-1C, as determined machinery. The load of proteins that cells process varies by western blot after treatment of the animal with the com considerably depending on the cell type and physiological pound. In addition, both PBA and TUDCA regulate insulin state of the cell. Cells can adapt by modulating the capacity of receptor signaling in animals, as measured by increased their ER to process proteins and the load of protein synthe tyrosine phosphorylation of insulin receptor, insulin substrate sized. Disequilibrium between ER load and folding capacity 1-IRS-1 and IRS-1, and increased serine phosphorylation of is referred to as ER stress (Harding et al. Diabetes 51 (Supp. Akt. TMAO has been shown to act as an anti-diabetic agent in 3):S455, 2002; incorporated herein by reference). ER stress vivo, lowering glucose and insulin levels when administered has been shown to be triggered by hypoxia, hypoglycemia, to animals with insulin resistance and type 2 diabetes. The exposure to natural toxins that perturb ER function, and a agent or a pharmaceutical composition of the agent is admin variety of mutations that affect the ability of client proteins to istered to a subject (e.g., human, dog, cat, mammal, animal) in fold (Lee, Trends Biochem. Sci. 26:504-510, 2001; Lee, Curr: doses effective to reduce ER stress, and thereby reduce the Opin. Cell Biol. 4:267-273, 1992; each of which is incorpo signs, symptoms, and consequences of obesity, insulin resis rated herein by reference). tance, and type 2 diabetes. The invention also provides meth 0005 Certain pathological conditions have been shown to ods of treating and/or preventing obesity, insulin resistance, disrupt ER homeostasis thereby leading to the accumulation type 2 diabetes, and hyperglycemia by administering agents of unfolded and misfolded proteins in the ER lumen (Hamp that reduce ER stress. The agents may be administered in any ton Curr. Biol. 10:R518, 2000; Mori Cell 101:451, 2000; manner known in the drug delivery art although preferably the Harding etal. Annu. Rev. Cell Dev. Biol. 18:575, 2002; each of agent is delivered orally or parenterally. Dose ranges for these US 2010/0075894 A1 Mar. 25, 2010

agents depend on the agent being delivered as well as other ful in the treatment of obesity, diabetes type 2, and insulin factors but will typically be from 10 mg/kg/day to 10 g/kg/ resistance (FIG. 7). TMAO, or a pharmaceutical composi day. tions thereof, is administered in doses ranging from 100 0007. In certain embodiments, the agent used to reduce ER mg/kg/day to 0.01 g/kg/day, preferably from 10 mg/kg/day to stress is 4-phenylbutyric acid (PBA). 0.1 g/kg/day, more preferably from 5 mg/kg/day to 0.5 mg/kg/day. 0010 Pharmaceutical compositions including agents that OH reduce ER stress and pharmaceutically acceptable excipients are also provided. The pharmaceutical compositions may be formulated for oral, parenteral, or transdermal delivery. The ER stress reducing agent may also be combined with other pharmaceutical agents. Such as insulin, anti-diabetics, PBA has been shown to regulate ER stress and regulate insu hypoglycemic agents, cholesterol lowering agents, appetite lin signaling. Phenylbutyric acid (PBA) or a derivative or salt Suppressants, aspirin, Vitamins, minerals, and anti-hyperten thereof is administered to a subject in order to reduce ER sive agents. For example, PBA may be combined with or stress and is particularly useful in the treatment of obesity, administered in conjunction with metformin. The agents may diabetes type 2, insulin resistance, and reducing blood glu be combined in the same pharmaceutical composition or may cose. PBA is effective in reducing blood glucose and increas be kept separate (i.e., in two separate formulations) and pro ing insulin sensitivity (FIGS. 4 and 5). PBA, or a pharmaceu vided together in a kit. The kit may also include instructions tical composition thereof, is administered in doses ranging for the physician and/or patient, Syringes, needles, box, from 10 mg/kg/day to 2 g/kg/day, preferably from 100 mg/kg/ bottles, vials, etc. day to 1 g/kg/day, more preferably from 500 mg/kg/day to 1 0011. In another aspect, the invention provides a method g/kg/day. of screening for agents that reduce ER stress. The identified 0008. In another embodiment, tauroursodeoxycholic acid agents are useful in the treatment of obesity, type 2 diabetes, (TUDCA), a bile acid, is the agent used to reduce ER stress. hyperglycemia, and insulin resistance. Agents to be screened are contacted with cells experiencing ER stress. The ER stress experienced by the cells may be caused by genetic alteration or treatment with a chemical compounds known to cause ER stress (e.g., tunicamycin, thapsigargin). Cells particularly useful in the inventive screen include liver cells and adipose cells. The levels of ER stress markers are then determined to identify agents that reduce ER stress. Examples of markers of ER stress include spliced forms of XBP-1, the phosphoryla tion status of PERK (Thr)80) and eIF2C. (Ser51), mRNA and protein levels of GRP78/BIP and JNK activity. Agents that when contacted with a cell with ER stress cause a reduction in the markers of ER stress as compared to an untreated control cell are identified as agents that reduce ER stress. A decrease in the levels of an ER stress marker are indicative of an agent TUDCA has been shown to regulate ER stress and insulin that is useful in treating diseases associated with ER stress, signaling. The invention provides the administration of taur Such as obesity, type 2 diabetes, insulin resistance, hypergly oursodeoxycholic acid (TUDCA) or a salt or derivative cemia, cystic fibrosis, and Alzheimer's diseases. Agents iden thereof to a subject in order to reduce ER stress. TUDCA has tified using the inventive method are part of the invention. been found to reduce blood glucose levels and increase insu These agents may be further tested for use in pharmaceutical lin sensitivity making it useful in the treatment of obesity, compositions. diabetes type 2, and insulin resistance (FIG. 6). TUDCA, or a 0012. In another aspect, the invention provides a method pharmaceutical composition thereof, is administered in doses of diagnosing insulin resistance, hyperglycemia, or type 2 ranging from 10 mg/kg/day to 2 g/kg/day, preferably from diabetes by measuring the level of expression of ER stress 100 mg/kg/day to 1 g/kg/day, more preferably from 250 markers. Markers which may be analyzed in the inventive mg/kg/day to 750 mg/kg/day. diagnostic method include spliced forms of XBP-1, phospho 0009. In another embodiment, TMAO is the agent used to rylation status of PERK, phosphorylation of eIF2C, mRNA reduce ER stress. levels of GRP78/BIP protein levels of GRP78/BIP and JNK activity. Any other cellular marker known to be indicative of ER stress may also be used. The levels of these markers may O be measured by any method known in the art including west N ern blot, northern blot, immunoassay, or enzyme assay. An HC1 YCH, increase in the level of an ER stress markers indicates that the CH3 Subject it at risk for insulin resistance, hyperglycemia, or type 2 diabetes.

TMAO has been shown to act as an anti-diabetic agent in vivo DEFINITIONS (see FIG. 7). The invention provides the administration of TMAO or a salt or derivative thereof to a subject in order to 0013 'Animal’: The term animal, as used herein, refers to reduce ER stress. TUDCA has been found to reduce blood humans as well as non-human animals, including, for glucose levels and increase insulin sensitivity making it use example, mammals, birds, reptiles, amphibians, and fish. US 2010/0075894 A1 Mar. 25, 2010

Preferably, the non-human animal is a mammal (e.g., a isofarnesyl group, a fatty acid group, a linker for conjugation, rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a functionalization, or other modification, etc. In a preferred primate, or a pig). In certain embodiments, the animal is a embodiment, the modifications of the peptide lead to a more human. stable peptide (e.g., greater half-life in vivo). These modifi 0014 “Chemical chaperone': A “chemical chaperone” is cations may include cyclization of the peptide, the incorpo a compound known to stabilize protein conformation against ration of D-amino acids, etc. None of the modifications denaturation (e.g., chemical denaturation, thermal denatur should substantially interfere with the desired biological ation), thereby preserving protein structure and function activity of the peptide. (Welch et al. Cell Stress Chaperones 1:109-115, 1996; incor (0017 “Polynucleotide' or “oligonucleotide” refers to a porated herein by reference). In certain embodiments, the polymer of nucleotides. The polymer may include natural “chemical chaperone' is a small molecule or low molecular nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, weight compound. Preferably, the “chemical chaperone' is uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, not a protein. Examples of “chemical chaperones include and deoxycytidine), nucleoside analogs (e.g., 2-thiothymi glycerol, deuterated water (DO), dimethylsulfoxide dine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-me (DMSO), trimethylamine N-oxide (TMAO), glycine betaine thylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluo (betaine), glycerolphosphocholine (GPC) (Burg et al. Am. J. rouridine, C5-iodouridine, C5-propynyl-uridine, Physiol. (Renal Physiol. 43): F762-F765, 1998; incorporated C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenos herein by reference), 4-phenylbutyrate or 4-phenylbutyric ine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, acid (PBA), methylamines, and taurourSodeoxycholic acid O(6)-methylguanine, 4-acetylcytidine, 5-(carboxyhy (TUDCA). Chemical chaperones may be used to influence droxymethyl)uridine, dihydrouridine, methylpseudouridine, the protein folding in a cell. Chemical chaperones have been 1-methyladenosine, 1-methylguanosine, N6-methyladenos shown in certain instances to correct folding/trafficking ine, and 2-thiocytidine), chemically modified bases, biologi defects seen in Such diseases as cystic fibrosis (Fischer et al. cally modified bases (e.g., methylated bases), intercalated Am. J. Physiol. Lung Cell Mal. Physiol. 281:L52-L57, 2001; bases, modified Sugars (e.g., fluororibose, ribose, 2'-deoxyri incorporated herein by reference), prion-associated diseases, bose. 2'-O-methylcytidine, arabinose, and hexose), or modi nephrogenic diabetes insipidus, and cancer (Baietal. Journal fied phosphate groups (e.g., phosphorothioates and 5'-N- of Pharmacological and Toxicological Methods 40(1):3945, phosphoramidite linkages). July 1998; incorporated herein by reference). Chemical chap 0018 “Small molecule': As used herein, the term “small erones also find use in the reduction of ER stress and are molecule' refers to organic compounds, whether naturally useful in the treatment of obesity, type 2 diabetes, insulin occurring or artificially created (e.g., via chemical synthesis) resistance, and hyperglycemia. that have relatively low molecular weight and that are not 0015 “Effective amount'. In general, the “effective proteins, polypeptides, or nucleic acids. Typically, Small mol amount of an active agent, such as an ER stress reducer or a ecules have a molecular weight of less than about 1500 g/mol. pharmaceutical composition thereof, refers to the amount Also, Small molecules typically have multiple carbon-carbon necessary to elicit the desired biological response. As will be bonds. Known naturally-occurring Small molecules include, appreciated by those of ordinary skill in this art, the effective but are not limited to, penicillin, erythromycin, taxol. amount of an agent that, reduces or prevents ER stress may cyclosporin, and rapamycin. Known synthetic Small mol vary depending on Such factors as the desired biological end ecules include, but are not limited to, amplicillin, methicillin, point, the agent being delivered, the disease being treated, the Sulfamethoxazole, and Sulfonamides. subject being treated, etc. For example, the effective amount of agent used to treat hyperglycemia or type 2 diabetes is the BRIEF DESCRIPTION OF THE DRAWINGS amount that results in a reduction in blood glucose levels by 0019 FIG. 1 evidences increased endoplasmic reticulum at least about 10%, 20%, 30%, 40%, or 50%. In other embodi stress seen in obesity. Dietary (high fat diet-induced) and ments, the effective amount of the ER stress modulator genetic (ob?ob) models of mouse obesity were used to exam reduces the levels of at least one ER stress marker (e.g., ine markers of ER stress in liver tissue compared to age and spliced forms of XBP-1, phosphorylation status of PERK, sex matched lean controls. ER stress markers including phosphorylation of eIF2C, mRNA levels of GRP78/BIP pro spliced forms of XBP-1 (XBP-1s), eIF2a phosphorylation tein levels of GRP78/BIP, and JNK activity). In certain (serS1, p-elF2a), PERK phosphorylation (p-PERK), mRNA embodiments, the levels of at least two, three, four, or more expression level of GRP78, and JNK activity were examined ER stress markers are reduced. The ER stress marker may be in the liver samples of the male mice (C57BL/6) that were reduced by approximately 10%, 20%, 30%, 40%, 50%, 60%, kept either on standard or high fat diet for 16 weeks. 100 ug of 70%, 80%, 90%, 95%, 98%, 99%, or 100%. protein were used for each immunoblot (FIG. 1a). Examina 0016 “Peptide' or “protein': According to the present tion of the same ER stress markers in the livers of male, ob?ob invention, a "peptide' or “protein’ comprises a string of at and wild type mice, between the ages of 12-14 weeks (FIG. least three amino acids linked together by peptide bonds. The 1b). Expression levels of GRP78 mRNA in liver were exam terms “protein’ and "peptide' may be used interchangeably. ined by Northern blot analyses in lean and obese animals Inventive peptides preferably contain only natural amino similar to the group described in FIGS. 1a and 1b (FIG. 1c). acids, although non-natural amino acids (i.e., compounds that 0020 FIG.2 shows how the induction of ER stress impairs do not occur in nature but that can be incorporated into a insulin action in liver cells via JNK-mediated phosphoryla polypeptide chain) and/oramino acid analogs as are known in tion of IRS-1. ER stress was induced in Fao cells, either with the art may alternatively be employed. Also, one or more of thapsigargin (thap, 300 nM for 4 hours) or tunicamycin (tun, the amino acids in an inventive peptide may be modified, for 10 ug/ml for 2 hours), and cells were Subsequently stimulated example, by the addition of a chemical entity Such as a car with insulin (ins). Insulin stimulated, IRS-1 tyrosine phos bohydrate group, a phosphate group, a farnesyl group, an phorylation and total protein levels in thapsigargin- or tuni US 2010/0075894 A1 Mar. 25, 2010 camycin-treated cells were examined after immunoprecipita tion) for 4 days, mice were injected intraperitoneally with tion (IP) of IRS-1 followed by immunoblotting (IB) with an TUDCA (500 mg/kg once a day in 200 ul of PBS) (n-6 for antibody against phospho-tyrosine (py) (a). Insulin stimu lean and n=7 for ob?ob) and PBS (200 ul) (n=6 lean and n=7 lated insulin receptor (IR) tyrosine phosphorylation and total for ob?ob). Blood glucose levels on the 4", 7", and 10" days protein levels in thapsigargin- or tunicamycin-treated cells after first injection (a). ITT on 10 days after the first injection (b). Phosphorylation of ser307 residue of IRS-1 in Fao cells (b). after 2 hours stimulation with thapSigargin (c). Inhibition of ser307 phosphorylation by JNK-1 inhibitor SP600 125 (0025 FIG. 7 shows the anti-diabetic effect of trimethy (JNKi) after thapsigargin treatment (d). Reversal of tunica lamine N-oxide (TMAO). Experiments were performed mycin-induced inhibition of insulin-stimulated tyrosine using the oblob genetic model of obesity and insulin resis phosphorylation (pY) of IRS-1 by blocking JNK activity with tance (C57BL/6J-Lep-ob mice purchased from Jackson a peptide inhibitor (JNKi). In these experiments, both immu Laboratory, Bar Harbor, Me...). At seven weeks of age, treat noprecipitation and immunoblotting were performed with ments were started after acclimatization for 5 days by admin anti-phosphotyrosine antibodies (e). istration of PBS. After this period, TMAO (Sigma, T0514) 0021 FIG.3 shows how IRE-1 plays a crucial role in ER was dissolved in PBS and administered by intraperitoneal stress mediated JNK activation, ser307 phosphorylation of (IP) injection every 24 hours at a dose of 1 g/kg/day. At the IRS-1, and inhibition of insulin receptor signaling. JNK activ indicated days, fed blood glucose levels were measured at ity was examined at indicated times following treatment with 8:00 AM using an automated glucometer system. For insulin tunicamycin in IRE1a+/+ and IRE-1a-/-fibroblasts using an measurements, blood samples were collected after 6 hours of in vivo kinase assay and recombinant c-jun as Substrate. JNK fasting and serum insulin levels were determined using a activity and total JNK levels (a). Phosphorylation of IRS-1 at specific ELISA (Crystal Chem, 90060). Each treatment group ser307 residue in IRE1a+/+ and IRE-1a-f- fibroblasts fol contained at least six animals per experimental group. lowing treatment with tunicamycin following IRS-1 immu 0026 FIG. 8 shows an effective combination of sodium noprecipitation (IP) and immunoblotting (IB) with an IRS-1 4-phenylbutyrate and metformin in the treatment of diabetes. phosphoserine 307-specific antibody (b). Insulin (ins)-stimu Experiments are performed using the ob?ob genetic model of lated IRS-1 tyrosine phosphorylation and total IRS-1 in levels obesity and insulin resistance (C57B6. V-Lepob/OlaHsd following treatment of IRE1a+/+ and IRE-1a-/- fibroblasts mice were purchased from Harlan Teklad, Madison, Wis.). At with tunicamycin. The graph below the blots shows the cor seven weeks of age, treatments were started after acclimati rected density of IRS-1 tyrosine phosphorylation to total zation for 5 days by administration of PBS. After this period, IRS-1 levels at each treatment time (c). PBS, sodium 4-phenylbutyrate (200 mg/kg/day), metformin 0022 FIG. 4 shows how the administration of 4-phenyl (200 mg/kg/day), and Sodium 4-phenylbutyrate plus met butyrate (4-PBA) via parenteral route increases insulin sen formin (200 mg/kg/day each) were administered into four sitivity in vivo and lowers blood glucose levels of diabetic separate experimental groups of mice (with at least six mice mice. A genetic (ob?ob) model of mouse obesity was used to in each group) by oral gavage daily. At these doses, only the analyze the effects of 4-phenylbutyrate (4-PBA) on insulin combined sodium 4-phenylbutyrate and metformin proved to sensitivity and hyperglycemia. 10-12 weeks old, male, leptin be effective as a blood glucose lowering regimen and single deficient mice were obtained from Jackson Labs, acclimated agents did not have any effect on blood glucose. Blood glu by three times injection of PBS (3 times a day) for 4 days, and cose levels were measured at the fed state at 8:00 AM. treated either with intraperitoneal injection of 4-PBA (1 g/kg/ (0027 FIG. 9 shows regulation of GRP78 expression by day in three divided doses) or phosphate buffer saline (PBS) glucose in vitro and hyperglycemia in vivo. (A) Fao cells were (3 doses of 100 ul) for a period of 7 days. Fed blood glucose treated with various doses of glucose (0, 5, 10, 25, and 75 levels (mg/dl) at the 3" and 7" days of trial (a). Insulin mM) for 24 hours. The mRNA level of GRP78 was examined Tolerance Test (ITT) were performed on the 7 day after the by Northern blot using the total RNAs isolated from these first injection (b). cells. Ethidium bromide staining is shown as a control for 0023 FIG. 5 shows how the administration of 4-phenyl loading and integrity of RNA. (B) Streptozotocin (STZ, 200 butyrate (4-PBA) via oral route increases insulin sensitivity in mg/kg) was injected intaperitoneally into male mice. Three vivo and lowers blood glucose levels of diabetic mice. A days after injection, blood glucose levels were measured to genetic (ob?ob) model of mouse obesity was used to analyze confirm STZ-induced hyperglycemia. Livers were collected the effects to 4-phenylbutyrate (4-PBA) on insulin sensitivity 10 days after injection and GRP78 expression was examined and hyperglycemia. 8-10 weeks old, male, leptin deficient by Northern blot analysis using the liver total RNA. mice were obtained from Jackson Labs, acclimated by PBS 0028 FIG. 10 shows ER stress indicators in adipose tis injection (2 times a day) for 4 days, and treated either with sues of obese mice. Dietary (high fat diet-induced) and 4-PBA (500 mg/kg/day in two divided doses) or phosphate genetic (ob?ob) models of mouse obesity were used to exam buffersaline (PBS) (2 doses of 200 ul) for a period of 20 days. ine markers of ER stress in adipose tissue compared with age Body weight during the treatment period (a). Blood glucose and sex matched lean controls. (A)PERK phosphorylation levels (mg/dl) after 6 hours of fasting at day 0 and day 20 (b). (p-PERK) and JNK activity were examined in the adipose Insulin (ng/ml) levels at day 0 and day 20 (c). Insulin toler samples of the male mice (C57BL/6) that were kept either on ance tests were performed at the 15" day of the treatment (d). standard diet (RD) or high fat diet (HFD) for 16 weeks. (B) 0024 FIG. 6 shows that the treatment of ob?ob mice with PERK phosphorylation and JNK activity in the adipose tis tauroursodexoycholic acid (TUDCA) increases insulin sen sues of male ob?ob and WT lean mice at the ages of 12-14 sitivity and reverses diabetes. 9-10 weeks old ob/ob and their weeks. (C) The mRNA levels of GRP78 were examined by age and sex matched lean controls were used to analyze the Northern blot analysis in the adipose tissues of WT lean and effects of TUDCA on glucose metabolism and diabetes. After ob?ob animals. Ethidium bromide staining is shown as a con acclimation with PBS injection (once a day, 200 ul i.p. injec trol for loading and integrity of RNA. US 2010/0075894 A1 Mar. 25, 2010

0029 FIG. 11 shows the induction of ER stress impairs phosphoserine 307-specific antibody. The graph next to the insulin action through JNK mediated phosphorylation of blots shows the quantitation of IRS-1 Ser307 phosphoryla IRS-1. (A) ER stress was induced in Fao liver cells by a tion under conditions described in panel E. (F) Insulin-stimu 3-hour treatment with 5ug/ml tunicamycin (Tun). Cells were lated tyrosine phosphorylation of IRS-1 in the XBP-1s over subsequently stimulated with insulin (Ins). IRS-1 tyrosine expressing and controls cells with or without tunicamycin and serine (Ser307) phosphorylation, Akt phosphorylation treatment (Tun, 2 ug/ml). The ratio of IRS-1 tyrosine phos (Ser473), insulin receptor (IR) tyrosine phosphorylation, and phorylation to total IRS-1 level was summarized from inde their total protein levels were examined using either immu pendent experiments and was presented in the graph. (G) noprecipitation (IP) followed by immunoblotting (IB) or IRS-1 Ser307 phosphorylation upon tunicamycin treatment direct immunoblotting. (B) Quantitation of IRS-1 (tyrosine (Tun, 0.5 g/ml) in XBP-1 cells and WT controls was and Ser307), Akt (Ser473), and IR (tyrosine) phosphorylation detected as described in panel C. The graph next to the blots under the experimental conditions described in (A) with nor shows the quantitation of IRS-1 Ser307 phosphorylation malization to protein levels for each molecule. (C) Inhibition under conditions described in FIG. 12G. (H) Insulin-stimu of ER stress-induced (300 nM thapsigargin for 4 hours) lated tyrosinephosphorylation of IRS-1 in XBP-1 and WT Ser307 phosphorylation of IRS-1 by JNK-1 inhibitor, control cells with or without tunicamycin treatment (Tun, 0.5 SP600 125 (JNKi, 25 uM), (D) Quantitation of IRS-1 Ser307 ug/ml). The ratio of IRS-1 tyrosine phosphorylation to total phosphorylation under conditions described in (C). (E) IRS-1 level was summarized from independent experiments Reversal of ER stress-induced inhibition of insulin-stimu and presented in the graph. All graphs show meantSEM from lated tyrosine phosphorylation (pY) of IRS-1 by a JNK at least 2 independent experiments and statistically signifi inhibitor. (F) Quantitation of insulin-induced IRS-1 tyrosine cance from the controls is indicated by * with p-0.005. phosphorylation levels described in (E). (G) JNK activity, 0032 FIG. 14 shows insulin-induced insulin receptor Ser307 phosphorylation of IRS-1, and total IRS-1 levels at autophosphorylation in XBP-1 overexpressing and XBP-1- indicated times following tunicamycin treatment (Tun, 10 deficient cells. (A) XBP-1 overexpressing cells and their con ug/ml for 1 hour) in IRE-1C.' and IRE-1C/ fibroblasts. (H) trol MEF cells (-Dox and +Dox, respectively) were treated Insulin-stimulated IRS-1 tyrosine phosphorylation and total with 2 ug/ml tunicamycin (Tun) for various period (0, 0.5, 1. IRS-1 levels following tunicamycin treatment (Tun, 10ug/ml 2, 3, and 4 hours). Insulin-induced insulin receptor (IR) for 1 hour) in IRE-1C." and IRE-1C fibroblasts. Quanti tyrosine phosphorylation (pY) and total IR levels were exam tation of insulin-induced IRS-1 tyrosine phosphorylation lev ined in those cells using immunoprecipitation (IP) with IR els in IRE-1C." and IRE-1C. cells is displayed in the bot antibody followed by immunoblotting (IB) with antibodies tom of the panel. All graphs show mean-SEM from at least 2 against IR orphospho tyrosine(py). (B) XBP-1 MEF cells independent experiments and Statistical significance from the and their WT controls were treated with 0.5ug/ml tunicamy controls is indicated by * with p<0.005. cin for various period (0, 0.5, 1, 2, 3, and 4 hours). Insulin 0030 FIG. 12 shows the inhibition of insulin receptor induced insulin receptor (IR) tyrosine phosphorylation (pY) signaling by thapsigargin-induced ER stress and the role of and total IR levels were examined as in panel A. Ca levels in IRS-1 serine phosphorylation. (A) ER stress was 0033 FIG. 15 shows glucose homeostasis in XBP-1 -- induced in Fao cells by 1 hour treatment with 300 nM thapsi mice on high fat diet. The XBP-1" (()) and XBP-1''' () gargin (Thap), and cells were Subsequently stimulated with mice were placed on high fat diet (HFD) immediately after insulin (Ins). IRS-1 tyrosine phosphorylation (pY) and serine weaning. Total body weight (A), fasting blood insulin (B), phosphorylation (pSer307), insulin receptor (IR) tyrosine C-peptide (C), and glucose (D) levels were measured in the phosphorylation, and total protein levels were examined XBP-1" and XBP-1" mice during the course of HFD. using either immunoprecipitation (IP) followed by immuno Glucose tolerance tests were performed after 7 (E) and 16 (F) blotting (IB) or direct immunoblotting. (B) Fao cells were weeks on HFD in XBP-1" and XBP-1" mice. Insulin tol treated with sulindac sulfide (SS: 0.7.5.30, and 60 uM) for 45 erance tests were performed after 8 (G) and 17 (H) weeks on minutes with or without an additional hour of exposure to 300 HFD in XBP-1" and XBP-1" mice. n=11 XBP-1" mice; nM thapSigargin (Thap). IRS-1 serine phosphorylation and n=8 XBP-1" mice. Data are shown as meaniSEM. Statisti total IRS-1 protein levels were examined as described above. cal significance in two-tailed student t test at ps0.05 is indi 0031 FIG. 13 demonstrates the alteration of the ER stress cated by *.ps0.005 by ** and ps0.0005 by ***. XBP-1" response by manipulation of XBP-1 levels leads to alterations and XBP-1" groups are also compared by ANOVA (panels in insulin receptor signaling. ER stress responses in XBP-1s A-H). overexpressing cells, XBP-1 cells and their controls. (A) 0034 FIG. 16 shows ER stress and insulin receptor sig Induction of XBP-1s expression upon removal of doxycy naling in XBP-1" mice. PERK phosphorylation (p-PERK) cline in mouse embryonic fibroblasts (MEF). (B) Southern (A), JNK activity (p-c-Jun) (B), and IRS-1 Ser307 (IRS blot analysis of XBP-1 MEF cells and their WT controls 1P-7) (C) were examined in the livers of XBP-1" and for the wild type (9.4 kb) and targeted (6.5 kb) alleles. (C) XBP-1" mice after 16 weeks on high fat diet. After infusion PERK phosphorylation (p-PERK) and JNK activity in the of insulin (1 U/kg) through portal vein, insulin receptor (IR) XBP-1s overexpressing cells and their control cells (-Dox tyrosine phosphorylation (pY) (D), IRS-1 tyrosine phospho and +Dox, respectively) upon tunicamycin treatment (Tun, 2 rylation (E), IRS-2 tyrosine phosphorylation (F), and Akt ug/ml). (D) PERK phosphorylation and JNK activity upon Ser473 phosphorylation (G) were examined in livers of XBP low dose tunicamycin treatment (Tun, 0.5ug/ml) in XBP-1 1" and XBP-1" mice after 16 weeks on high fat diet. MEF cells and their WT controls. (E) IRS-1 Ser307 phospho 0035 FIG. 17 is the characterization of pancreatic islets in rylation upon tunicamycin treatment (Tun, 2 ug/ml) in the XBP-1" and XBP-1" mice. Islet morphology, size, and XBP-1s overexpressing and the control cells (-Dox and immunohistochemical staining for insulin and glucagon in +DOX, respectively), detected using immunoprecipitation pancreatic sections obtained from XBP-1" and XBP' (IP) of IRS-1 followed by immunoblotting (IB) with an IRS-1 mice on either regular diet (A-D) or HFD (E-H). Glucose US 2010/0075894 A1 Mar. 25, 2010 stimulated insulin secretion in XBP-1" and XBP-1" mice "Endoplasmic Reticulum Stress Link Obesity, Insulin Action, on high fat diet Glucose-stimulated insulin secretion was and Type 2 Diabetes’ Science 306:457-461, 2004; incorpo examined in XBP-1" and WT mice placed on high fat diet rated herein by reference). Based on this discovery, agents for 16 weeks (I). Glucose was administered introperitoneally that reduce or prevent ER stress have been shown to be useful to mice in each genotype and blood samples are collected at in the treatment of obesity, insulin resistance, hyperglycemia, the indicated times for insulin measurements. In these experi and type 2 diabetes. ments, there was no detectable abnormality in the XBP" 0041 Any agent known to reduce or modulate ER stress is islets and no difference was evident between genotypes under useful in treating these metabolic diseases. These agents may standard conditions. On HFD, both the XBP-1" and XBP act to reduce or prevent ER stress in any manner. In certain 1" mice exhibited islet hyperplasia. This anticipated embodiments, the agent may increase the capacity of the ER response to HFD was similar between genotypes and the to process proteins (e.g., increasing the expression of ER hyperplastic component (islet size >150 uM) comprised 40% chaperones, increasing the levels of post-translational of all islets in XBP-1" and 43% of all islets in WT mice on machinery). In other embodiments, the agent may reduce the HFD. In experiments examining glucose-stimulated insulin quantity of proteins to be processed by the ER (e.g., decreas secretion in XBP-1" and WT mice on HFD, the XBP-1" ing the total level of protein produced in a cell, reducing the mice responded to glucose with even a stronger insulin secre level of protein processed by the ER, reducing the level of tory response, which effectively eliminates the possibility of mutant proteins, reducing the level of misfolded proteins). Yet an isolated islet defect underlying their phenotype. Hence, other agents may cause the release of misfolded/mutant pro these data indicate that the phenotype of the XBP-1" mice teins from the ER. The agent may work in all cells, or the cannot be explained by defective islets and even after 16 effect may be limited to certain cells type (e.g., secretory weeks on HFD, the islets appear indistinguishable between cells, epithelial cells, hepatocytes, adipocytes, endocrine genotypes. cells, etc.). In certain embodiments, the agents are particu 0036 FIG. 18 shows ER stress and insulin receptor sig larly useful in reducing ER stress in adipose cells. In other naling in XBP-1" mice. PERK phosphorylation (p-PERK) embodiments, the agents are particularly useful in reducing (A), JNK activity (p-c-Jun) (B), and IRS-1 Ser307 (IRS ER stress in hepatic cells. The agents may work on the tran 1') (C) were examined in the livers of XBP-1" and Scriptional, translational, post-translational, or protein level XBP-1" mice after 16 weeks on high fat diet. After infusion to reduce or prevent ER stress. of insulin (1 U/kg) through portal vein, insulin receptor (IR) 0042. The administration of an effective dose of an ER tyrosine phosphorylation (pY) (D), IRS-1 tyrosine phospho stress modulator, or a combination therapy including an ER rylation (E), IRS-2 tyrosine phosphorylation (F), and Akt stress modulator, to a Subject to treat or prevent obesity, Ser473 phosphorylation (G) were examined in livers of XBP insulin resistance, type 2 diabetes, hyperglycemia, or other 1" and XBP-1" mice after 16 weeks on high fat diet. related disease may cure the disease being treated, alleviate or 0037 FIG. 19 shows intact insulin receptor signaling in reduce at least one sign or symptoms of the disease being liver and adipose tissues of XBP-1" and XBP-1" mice on treated, reduce the short term consequences of the disease, regular diet. After infusion of insulin (1 U/kg) through the reduce the long term consequences of the disease, or provide portal vein, insulin receptor (IR) tyrosine phosphorylation some other transient beneficial effect to the subject. In certain (pY), IRS-1 tyrosine phosphorylation, IRS-2 tyrosine phos embodiments, the inventive treatment increases insulin sen phorylation, Akt Ser473 phosphorylation, and their total pro sitivity. In other embodiments, the inventive treatment tein levels were examined in livers (A) and adipose tissues (B) decreases blood glucose levels. In other embodiments, the of XBP-1" and XBP' mice on regular diet. inventive treatment prevents the long term consequences of 0038 FIG. 20 shows reduced insulin receptor signaling in diabetes including atherosclerosis, diabetic retinopathy, adipose tissues of XBP-1" and XBP-1" mice on high fat peripheral neuropathy, etc. In certain embodiments, the diet. (A) After infusion of insulin (1 U/kg) through the portal inventive treatment reduces levels of ER stress markers (e.g., vein, insulin receptor (IR) tyrosine phosphorylation (pY), spliced froms of XBP-1, phosphorylation status of PERK, IRS-1 tyrosine phosphorylation, IRS-2 tyrosine phosphory phosphorylation of eIF2C, mRNA levels of GRP78/BIP pro lation, Akt Ser473 phosphorylation, and their total protein tein levels of GRP78/BIP JNK activity) in cells (e.g., adipo levels were examined in adipose tissues of XBP-1" and cytes, hepatocytes). In certain embodiments, the inventive XBP' mice on high fat diet for 16 weeks. (B) JNK kinase treatment increases insulin action. In other embodiments, the assay was performed in adipose tissues of XBP-1" and inventive treatment increases insulin receptor signalling (e.g., XBP' mice on high fat diet for 16 weeks. phosphorylation of insulin receptor, IRS-1, IRS-2, akt). In 0039 FIG. 21 shows increased JNK activity in the liver certain embodiments, the inventive treatment Suppresses tissue of obese mice followed by normalization of JNK activ appetite. In other embodiments, the inventive treatment pre ity after treatment with PBA. vents weight gain or promotes weight loss. In certain embodi ments, the inventive treatment prevents the development of DETAILED DESCRIPTION OF CERTAIN type 2 diabetes. In certain embodiments, the inventive treat PREFERRED EMBODIMENTS OF THE ment prevents the development of obesity. In certain embodi INVENTION ments, the inventive treatment prevents the development of 0040 Endoplasmic reticulum (ER) stress has been found hyperglycemia. to be important in the pathogenesis of a variety of diseases 0043. The agent may be any type of chemical compound. including C.1-anti-trypsin deficiency, urea cycle disorders, The agent may be a small molecule, organometallic complex, type 1 diabetes, and cystic fibrosis. The present invention an inorganic compound, a protein, a glycoprotein, a peptide, stem from the recognition that ER stress is implicated in the a carbohydrate, a lipid, or a nucleic acid. In certain embodi pathogenesis of diseases Such as obesity, peripheral insulin ments, the agent is a small molecule. Particularly useful resistance, hyperglycemia, and type 2 diabetes (OZcan et al., agents are known as chemical chaperones, which are known US 2010/0075894 A1 Mar. 25, 2010 to stabilize proteins against denaturation thereby preserving thereof) is used in combination with an anti-obesity agent. the protein's structure and function. Chemical chaperones Exemplary anti-obesity agents include pancreatic lipase include glycerol, DO, dimethylsulfoxide (DMSO), 4-phenyl inhibitors (e.g., orlistat), serotonin and norepinephrine butyrate (PBA), tauroursodeoxycholic acid (TUDCA), gly reuptake inhibitors (e.g., Sibutramine), noradrenergic anorec cine betaine (betaine), glycerolphosphocholine (GPC), tic agents (e.g., phentermine, mazindol), peripherally acting methylamines, and trimethylamine N-oxide (TMAO). In cer agents (e.g., ATL-962 (Alizyme), HMR-1426 (Aventis), tain embodiments, combinations of one or more chemical GI-181771 (GlaxoSmithKline)), centrally acting agents (e.g., chaperones may be used. These chemical chaperones are Recombinant human ciliary neurotrophic factor, Rimonabant administered in doses ranging from 10 mg/kg/day to 10 g/kg/ (SR-141716) (Sanofi-Synthélabo), BVT-933 (GlaxoSmith day, preferably 100 mg/kg/day to 5 g/kg/day, more preferably Kline/Biovitrum), Bupropion SR (GlaxoSmithKline), P-57 from 500 mg/kg/day to 3 g/kg/day. In certain embodiments, (Phytopharm)), thermogenic agents (e.g., TAK-677 (AJ the agent is administered in divided doses (e.g., twice per day, 9677) (Dainippon/Takeda)), cannabinoid CB1 antagonists three times a day, four times a day, five times a day). In other (e.g., acomplia, SLV319), cholecystokinin (CCK) agonists embodiments, the agent is administered in a single dose per (e.g., GI 181771 (GSK)), lipid metabolism modulator (e.g., day. AOD9604 (Monash University/Metabolic Pharmaceuticals), 0044) The agent may be combined with one or more other glucagon-like peptide 1 agonist (e.g., AC137 (Amylin)), lep pharmaceutical agents, particularly agents traditionally used tin agonist (e.g., second generation leptin (Amgen), beta-3 in the treatment of diabetes, obesity, or insulin resistance. A adrenergic agonists (e.g., SR58611 (Sanofi-Aventis), CP list of agents useful in combination with ER stress modulators 33 1684 (Pfizer), LY 377604 (Eli Lilly), n5984 (Nisshin (e.g., PBA, TUDCA, TMAO, or derivatives thereof) is Kyorin Pharmaceutical)), peptide hormone (e.g., peptide YY included as Appendix A. The list includes generic names, 3-36 (Nastech)), CNS modulator (e.g., S2367 (Shionogi & trade names, and manufacturers. Exemplary agents useful in Co. Ltd.)), neurotrophic factor (e.g., peg axokine), and combination with ER stress reducing agents include, but are 5HT2C serotonin receptor agonist (e.g., APD356). Other not limited to, anti-diabetic agents (e.g., insulin, hypoglyce anti-obesity agents include methamphetamine HCl, 1426 mic agents (e.g., oral hypoglycemic agents such as Sulfony (Sanofi-Aventis), 1954 (Sanofi-Aventis), c-2624 (Merck & lureas, tolbutamide, metformin, chlorpropamide, acetohexa Co), c-5093 (Merck & Co), and T71 (Tularik). mide, tolaZamide, glyburide, etc.)), anti-obesity agents, anti 0047. In yet other embodiments, a chemical chaperone or dyslipidemia agent or anti-atherosclerosis agent (e.g., ER stress modulator (e.g., PBA, TUDCA, TMAO, or deriva cholesterol lowering agents (e.g., HMg-CoA reductase tives thereof) is used in combination with an anti-dyslipi inhibitors such as lovastatin, atorvastatin, simvastatin, prav demia agent or anti-atherosclerosis agent. Exemplary anti astatin, fluvastatin, etc., aspirin), anti-obesity agent (e.g., dyslipidemia agents or anti-atherosclerosis agents include appetite Suppressants), vitamins, minerals, and anti-hyper HMG-CoA reductase inhibitors (e.g., atorvastatin, pravasta tensive agents. tin, simvastatin, lovastatin, fluvastatin, cerivastatina, rosuv 0045. In certain embodiments, a chemical chaperone or astatin, pitivastatin), fibrates (e.g., ciprofibrate, beZafibrate, ER stress modulator (e.g., PBA, TUDCA, TMAO, or deriva clofibrate, fenofibrate, gemfibrozil), bile acid sequestrants tives thereof) is used in combination with an anti-diabetic (e.g., cholestyramine, colestipol, colesevelam), niacin (im agent. Examplary anti-diabetic agents include biguanides mediate and extended release), anti-platelets (e.g., aspirin, (e.g., metformin), Sulfonylureas (e.g., glimepiride, glyburide, clopidogrel, ticlopidine), angiotensin-converting enzyme glibenclamide, glipizide, gliclazide), insulin and analogs (ACE) inhibitors (e.g., ramipril, enalapril), angiotensin II thereof (e.g., insulin lispro, insulin glargine, exubera, AERX receptor antagonists (e.g., losartan potassium), acyl-CoA insulin diabetes management system, AIR inhaled insulin, cholesterol acetyltransferase (ACAT) inhibitors (e.g., ava oralin, insulin detemir, insulin glulisine), peroxisome prolif simibe, eflucimibe, CS-505 (Sankyo and Kyoto), SMP-797 erator-activated receptor-gamma agonists (e.g., rosiglita (Sumito)), cholesterol absorption inhibitors (e.g., eZetimibe, Zone, pioglitaZone, isaglitaZone, rivoglitaZone, T-131, MBX pamaqueside), nicotinic acid derivatives (e.g., nicotinic acid), 102, R-483 CLX-0921), dual PPAR agonists and PPAR pan cholesterol ester transfer protein (CETP) inhibitors (e.g., agonists (e.g., BMS-398585, tesaglitazar, muraglitazar, CP-529414 (Pfizer), JTT-705 (Japan Tobacco), CETi-1, naveglitazar, TAK-559, netoglitazone, GW-677594, AVE torcetrapib), microsomal triglyceride transfer protein 0847, LY-929, ONO-5129), combination therapies (e.g., met (MTTP) inhibitors (e.g., implitapide, R-103757, CP-346086 formin/glyburide, metformin/rosiglitaZone, metformin, glip (Pfizer)), other cholesterol modulators (e.g., NO-1886 (Ot izide), meglitinides (e.g., repaglinide, nateglinide), alpha suka/TAP Pharmaceutical), CI-1027 (Pfizer), WAY-135433 glucosidase inhibitors (e.g., acarbose, miglitol, Voglibose), (Wyeth-Ayerst)), bile acid modulators (e.g., GT102-279 glucagon-like peptide-1 (GLP-1) analogues and agonists (GelTex/Sankyo), HBS-107 (Hisamitsu/Banyu), BTG-511 (e.g., Exenatide, Exenatide LAR, Liraglutide, CJC-1131, (British Technology Group), BARI-1453 (Aventis), S-8921 AVE-0010, BIM-51077, NN-2501, SUN-E7001), dipeptidyl (Shionogi), SD-5613 (Pfizer), AZD-7806 (AstraZeneca)), peptidase IV (DPP-IV) inhibitors (e.g., LAF-237, MK-431 peroxisome proliferation activated receptor (PPAR) agonists (Merck and Co), PSN-9301 (Probiodrug Prosidion), 815541 (e.g., Tesaglitazar (AZ-242) (AstraZeneca), NetoglitaZone (GlaxoSmithKline-Tanabe), 823.093 (GlaxoSmithKline), (MCC-555) (Mitsubishi/Johnson & Johnson), GW-409544 825964 (GlaxoSmithKline), BMS-477118), pancreatic (Ligand Pharmaceuticals/GlaxoSmithKline), GW-501516 lipase inhibitors (e.g., orlistat), Sodium glucose co-trans (Ligand Pharmaceuticals/GlaxoSmithKline), LY-929 porter (SGLT) inhibitors (e.g., T-1095 (Tanabe-J&J), AVE (Ligand Pharmaceuticals and Eli Lilly), LY-465608 (Ligand 2268, 869682 (GlaxoSmithKline-Kissei)), and amylin ana Pharmaceuticals and Eli Lilly), LY-518674(Ligand Pharma log (e.g., pramlintide). ceuticals and Eli Lilly), MK-767 (Merck and Kyorin)), gene 0046. In other embodiments, a chemical chaperone or ER based therapies (e.g., AdGVVEGF121.10 (GenVec), ApoA1 stress modulator (e.g., PBA, TUDCA, TMAO, or derivatives (UCB Pharma/Groupe Fournier), EG-004 (Trinam) (Ark US 2010/0075894 A1 Mar. 25, 2010

Therapeutics), ATP-binding cassette transporter-A1 hydrochlorothiazide, lisinopril/hydrochlorothiazide, (ABCA1) (CV. Therapeutics/Incyte, Aventis, Xenon)), com losartan/hydrochlorothiazide, atenolol/chlorthalidone, biso posite vascular protectant (e.g. AGI-1067 (Atherogenics)), prolol fumarate/hydrochlorothiazide, metoprolol tartrate/hy BO-653 (Chugai), glycoprotein IIb/IIIa inhibitors (e.g., Roxi drochlorothiazide, amlodipine besylate/benazepril hydro fiban (Bristol-Myers Squibb), Gantofiban (Yamanouchi), chloride, felodipine?enalapril maleate, Verapamil Cromafiban (Millennium Pharmaceuticals)), aspirin and ana hydrochloride/trandolapril, lercanidipine and enalapril, olm logs thereof (e.g., asacard, slow-release aspirin, pamicogrel), esartan/hydrochlorothiazide, eprosartan/hydrochlorothiaz combination therapies (e.g., niacin/lovastatin, amlodipine/ ide, amlodipine besylate/atorvastatin, nitrendipine?enala atorvastatin, simvastatin/ezetimibe), IBAT inhibitors (e.g., pril), and MC4232 (University of Manitoba/Medicure). S-89-21 (Shionogi)), squalene synthase inhibitors (e.g., 0049. In certain embodiments, a chemical chaperone or BMS-188494. I(Bristol-Myers Squibb), CP-210172 (Pfizer), ER stress modulator (e.g., PBA, TUDCA, TMAO, or deriva CP-295697 (Pfizer), CP-294.838 (Pfizer), TAK-475 tives thereof) is used in combination with a vitamin, mineral, (Takeda)), monocyte chemoattractant protein (MCP-1) or other nutritional Supplement. inhibitors (e.g., RS-504393 (Roche Bioscience), other 0050. In certain embodiments, the ER stress modulator MCP-1 inhibitors (GlaxoSmithKline, Teijin, and Bristol-My (e.g., PBA, TUDCA, TMAO, orderivatives thereof) is admin ers Squibb)), liver X receptor agonists (e.g., GW-3965 istered in a Sub-optimal dose (e.g., an amount that does not (GlaxoSmithKline), TU-0901317 (Tularik)), and other new manifest detectable therapeutic benefits when administered approaches (e.g., MBX-102 (Metabolex), NO-1886 (Ot in the absence of a second agent). In Such cases, the admin suka), Gemeabene (Pfizer)). istration of such an sub-optimal dose of the ER stress modu 0048. In still other embodiments, a chemical chaperone or lator in combination with another agent results in a synergis ER stress modulator (e.g., PBA, TUDCA, TMAO, or deriva tic effect. The ER stress modulator and other agent work tives thereof) is used in combination with an anti-hyperten together to produce a therapeutic benefit. In other embodi sive agent. Examplary anti-hypertension agents include ments, the other agent (i.e., not the ER stress modulator) is diurectics (e.g., chlorthalidone, metolaZone, indapamide, administered in Sub-optimal doses. In combination with an bumetanide, ethacrynic acid, furosemide, torsemide, ER stress modulator, the combination exhibits a therapeutic amiloride HCl, spironolactone, triamterene), alpha-blockers effect. In yet other embodiments, both the ER stress modula (e.g., doxazosin mesylate, praZosin HCl, teraZosin HCl). tor and the other agent are administered in Sub-therapeutic beta-blockers (e.g., acebutolol, atenolol betaxolol, bisoprolol doses, and when combined produce a therapeutic effect. The fumarate, carteolol HCl, metoprolol tartrate, metoprolol suc dosages of the other agent may be below those standardly cinate, nadolol, penbutolol Sulfate, pindolol, propanolol HCl, used in the art. timolol maleate, carvedilol), Ca" channel blockers (e.g., 0051. The dosages, route of administration, formulation, amlodipine besylate, felodipine, isradipine, nicardipine, nife etc. for anti-diabetic agents, anti-obesity agents, anti-dyslipi dipine, nisoldipine, diltiazem HCl, Verapamil HCl, azelnid demia agent or anti-atherosclerosis agent, anti-obesity agent, ipine, pranidipine, graded diltiazem formulation, (S)-amlo Vitamins, minerals, and anti-hypertensive agents (listed dipine, clevidipine), angiotensin converting enzyme (ACE) above) are known in the art. The treating physician or health inhibitors (e.g., benazepril hydrochloride, captopril, enalapril care professional may consult Such references as the Physi maleate, fosinopril Sodium, lisinopril, moexipril, perindopril, cian's Desk Reference (59" Ed., 2005), or Mosby's Drug quinapril hydrochloride, ramipril, trandolapril), angiotensin Consult and Interactions (2005) for such information. It is II (AT-II) antagonists (e.g., losartan, Valsartan, irbesartan, understood that a treating physician would exercise his pro candesartan, telmisartan, eprosartan, olmesarta, YM-358 fessional judgment to determine the dosage regimen for a (Yamanouchi)), Vasopeptidase inhibitors (e.g. omapatrilat, particular patient. gemopatrilat, fasidotril, sampatrilat, AVE 7688 (Aventis), 0.052 The invention provides systems and methods of M100240 (Aventis), Z13752A (Zambon/GSK), 796406 treating type 2 diabetes, insulin resistance, obesity, and other (Zambon/GSK)), dual neutral endopeptidase and enotheline related conditions that provide a better therapeutic profile converting enzyme (NEP/ECE) inhibitors (e.g., SLV306 than the administration of the ER stress modality or the other (Solvay), NEP inhibitors (e.g., ecadotril), aldosterone treatment modality alone. In certain embodiments, the thera antagonists (e.g., eplerenone), renin inhibitors (e.g., Aliskiren peutic effect may be greater. In certain embodiments, the (Novartis), SPP 500 (Roche/Speedel), SPP600 (Speedel), combination has a synergistic effect. In other embodiments, SPP800 (Locus/Speedel)), angiotensin vaccines (e.g., PMD the combination has an additive effect. The administration of 3117 (Protherics)), ACE/NEP inhibitors (e.g., AVE-7688 a combination treatment regimen may reduce or even avoid (Aventis), GW-660511 (Zambon SpA)), Na"/K"ATPase certain unwanted or adverse side effects. In certain embodi modulators (e.g., PST-2238 (Prassis-Sigma-Tau), endothelin ments, the agents in the combination may be adminstered in antagonists (e.g., PD-156707 (Pfizer)), vasodilators (e.g., lowerdoses, adminstered less frequently, or administered less NCX-4016 (NicOx), LP-805 (Pola/Wyeth)), naturetic pep frequently and in lower doses. Therefore, combination thera tides (e.g., BDNP (Mayo Foundation)), angiotensin receptor pies with the above described benefits may increase patient blockers (ARBs) (e.g., pratosartan), ACE crosslink breakers compliance, improve therapy, and/or reduce unwanted or (e.g., alagebrium chloride), endothelin receptor agonists adverse side effects. (e.g., tezosentan (Genentech), ambrisentan (Myogen), 0053. In certain embodiments, a chemical chaperone (e.g., BMS193884 (BMS), sitaxsentan (Encysive Pharmaceuti PBA, TUDCA, TMAO, or derivatives thereof) is used in cals), SPP301 (Roche/Speedel), Darusentan (Myogen/Ab combination with a hypoglycemic agent. For example, insu bott), J104132 (Banyu/Merck & Co.), TBC37.11 (Encysive lin, glucagon, a biguanide hypoglycemic agent (e.g., met Pharmaceuticals), SB 234551 (GSK/Shionogi)), combina formin, phenformin, or buformin), a thiazolidinedione tion therapies (e.g., benazepril hydrochloride/hydrochlo hypoglycemic agent (e.g., ciglitaZone, pioglitaZone), a Sulfo rothiazide, captopril/hydrochlorothiazide, enalapril maleate/ nylurea hypoglycemic agent (e.g., tolbutamide, chlorpropa US 2010/0075894 A1 Mar. 25, 2010

mide, acetohexamide, tolaZamide, glyburide, glipizide, or monosubstituted phenyl ring. In yet other embodiments, Rois gliclazide), an O-glucosidase inhibitor (e.g., acarbose), or a disubstituted phenyl ring. In still other embodiments, Ro is diazoxide may be combined with glycerol, DO, dimethyl a trisubstituted phenyl ring. In certain embodiments, Ro is a sulfoxide (DMSO), 4-phenyl butyrate (PBA), tauroursode phenyl ring Substituted with 1, 2, 3, or 4 halogen atoms. In oxycholic acid (TUDCA), glycine betaine (betaine), glycer certain embodiments, Ro is a Substituted or unsubstituted olphosphocholine (GPC), methylamines, or trimethylamine heteroaryl ring. In certain embodiments, Ro is a naphthyl ring. N-oxide (TMAO). Certain specific exemplary combination In certain embodiments, Ro is five- or six-membered, prefer therapies include insulin and PBA, insulin and TUDCA, insu ably six-membered. In certain embodiments, R and R2 are lin and betaine, insulin and GPC, insulin and TMAO, met both hydrogen. In certain embodiments, n is 1. In other formin and PBA, metformin and TUDCA, metformin and embodiments, n is 2. In certain embodiments, all R and R. betaine, metformin and GPC, metformin and TMAO, a thia are hydrogen. In other embodiments, at least one R or R is Zolidinedione hypoglycemic agent and PBA, a thiazo hydrogen. In certain embodiments, the compound is used in a lidinedione hypoglycemic agent and TUDCA, a thiazo salt form (e.g., sodium salt, potassium salt, magnesium salt, lidinedione hypoglycemic agent and betaine, a ammonium salt, etc.) Other derivatives useful in the present thiazolidinedione hypoglycemic agent and GPC, and a thia invention are described in U.S. Pat. No. 5,710,178, which is Zolidinedione hypoglycemic agent and TMAO. In certain incorporated herein by reference. 4-phenyl butyrate or its embodiments, the combination used to treat or prevent obe derivatives may be obtained from commercial sources, or sity, insulin resistance hyperglycemia, or type 2 diabetes is prepared by total synthesis or semi-synthesis. 4-phenylbutyrate (PBA) and metformin. In terms of combi 0060. In certain embodiments, a derivative of TUDCA nation therapies whether they be combinations of chemical useful in the present invention is of the formula: chaperones, combinations of ER stress modulators, or com binations of chemical chaperones/ER stress modulators and other agents such as hypoglycemic agents, the agents may be delivered concurrently or consecutively. In certain embodi CH ments, the chemical chaperone or ER stress modulator is O administered before the other agent. In other embodiments, the chemical chaperone or ER stress modulator is adminis CH tered after the other agent. RN 0054) In certain embodiments, small molecule agents shown to reduce ER stress include 4-phenylbutyrate (PBA), )-R, tauroursodeoxycholic acid (TUDCA), and trimethylamine HO OH R N-oxide (TMAO). PBA is used currently to treat O. 1-anti trypsin deficiency, urea cycle disorders, and cystic fibrosis. wherein: Derivatives, salts (e.g., Sodium, magnesium, potassium, mag 0061 R is —H or C-C alkyl; nesium, ammonium, etc.), prodrugs, esters, isomers, and Ste 0062 R is —CH2—SOR and R is —H; or R is reoisomers of PBA, TUDCA, or TMAO may also be used to —COOH and R is —CH, CH CONH —CH2— treat obesity, hypergylcemia, type 2 diabetes, and insulin CONH2, —CH2—CH2—SCH or —C. S CH resistance. Without wishing to be bound by any particular COOH; and theory, these compounds are thought to work by allowing the 0.063 R is —H or a basic amino acid; or a pharmaceu ER to better handle misfolded and/or mutant proteins being tically acceptable salt thereof. In certain embodiments, processed by the ER. the stereochemistry of the derivative is defined as shown 0055. In certain embodiments, a derivative of 4-phenyl in the following structure: butyrate useful in the present invention is of the formula:

R R3 O R- OH R0 R4 pi wherein n is 1 or 2; 0056 Ro is aryl, heteroaryl, or phenoxy, wherein the aryl, H heteroaryl, and phenoxy being unsubstituted or Substituted with, independently, one or more halogen, hydroxy, or lower In certain embodiments, R is H. In other embodiments, R is alkyl (C-C) groups; methyl, ethyl, n-propyl, iso-propyl. n-butyl, iso-butyl, or tort 0057 R and R are independently H. lower alkoxy, butyl, preferably, methyl. In certain embodiments, R or R is hydroxy, lower alkyl or halogen; and hydrogen. In certain embodiments, R is —CH2—SOR and 0058 R and Ra are independently H. lower alkyl, lower R is —H. In other embodiments, R is COOH and R is alkoxy or halogen; or —CH, CH, CONH, —CH CONH, -CH 0059 a pharmaceutically-acceptable salt thereof; or a CH SCH or —CH2—S CH-COOH. In certain mixture thereof. In certain embodiments, Ro is a substituted embodiments, R is hydrogen. In certain embodiments, R3 is or unsubstituted phenyl ring. In certain embodiments, Roisan lysine, arginine, ornithine, or histidine. Derivatives of unsubstituted phenyl ring. In other embodiments, Ro is a TUDCA and ursodeoxycholic acid may be obtained from US 2010/0075894 A1 Mar. 25, 2010 commercial sources, prepared from total synthesis, or include a pharmaceutically acceptable excipient or carrier. As obtained from a semi-synthesis. In certain embodiments, the used herein, the term “pharmaceutically acceptable carrier' derivative is prepared via semi-synthesis, for example, as means a non-toxic, inert Solid, semi-solid or liquid filler, described in U.S. Pat. Nos. 5,550,421 and 4,865,765, each of diluent, encapsulating material, or formulation auxiliary of which is incorporated herein by reference. any type. Some examples of materials which can serve as 0064. In certain embodiments, derivative of trimethy pharmaceutically acceptable carriers are Sugars such as lac lamine N-oxide useful in the present invention is of the for tose, glucose, and Sucrose; starches Such as corn starch and mula: potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt, gelatin; talc, excipients Such as cocoa butter and Suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil, olive oil; corn oil; and Soybean oil; glycols such as propylene glycol, esters such as ethyl oleate and ethyl laurate; agar; detergents Such as Tween 80; buffering agents such as magnesium wherein hydroxide and aluminum hydroxide; alginic acid; pyrogen 0065 R. R. and R are independently hydrogen, halo free water, isotonic saline; Ringer's Solution; ethyl alcohol; gen, or lower C-C alkyl; or artificial cerebral spinal fluid (CSF), and phosphate buffer 0066 a pharmaceutically-acceptable salt thereof; or a Solutions, as well as other non-toxic compatible lubricants mixture thereof. In certain embodiments, R, R2, and R are Such as sodium lauryl Sulfate and magnesium Stearate, as well the same. In other embodiments, at least one of R, R2, and R as coloring agents, releasing agents, coating agents, Sweeten is different. In yet other embodiments, all of R. R., and R. ing, flavoring, and perfuming agents, preservatives and anti are different. In certain embodiments, R, R2, and R are oxidants can also be present in the composition, according to independently hydrogen or lower C-C alkyl. In yet other the judgment of the formulator. The pharmaceutical compo embodiments, R. R. and R are independently lower C-C, sitions of this invention can be administered to humans and/or alkyl. In still other embodiments, R, R2, and R are indepen to animals, orally, rectally, parenterally, intracisternally, dently methyl, ethyl, or propyl. In certain embodiments, R. intravaginally, intranasally, intraperitoneally, topically (as by R, and R are ethyl. Derivatives of TMAO may be obtained powders, creams, ointments, or drops), transdermally, Subcu from commercial Sources, or prepared by total synthesis or taneously, bucally, or as an oral or nasal spray. semi-synthesis. 0071 Injectable preparations, for example, sterile inject 0067. In other embodiments, the agent is a nucleic acid, able aqueous or oleaginous Suspensions may be formulated e.g., an inhibitory RNA such as an siRNA. In other embodi according to the known art using Suitable dispersing or wet ments, the agent is a protein, e.g., an antibody or antibody ting agents and Suspending agents. The sterile injectable fragment. In yet other embodiments, the agent is a peptide. preparation may also be a sterile injectable solution, Suspen 0068. In treating an animal, suffering from obesity, Sion, or emulsion in a nontoxic parenterally acceptable dilu peripheral insulin resistance, hyperglycemia, or type 2 diabe ent or solvent, for example, as a solution in 1,3-butanediol. tes, a therapeutically effective amount of the agent is admin Among the acceptable vehicles and solvents that may be istered to the subject via any route to achieve the desired employed are water, Ringer's solution, U.S.P. and isotonic biological result. Any route of administration may be used sodium chloride solution. In addition, sterile, fixed oils are including orally, parenterally, intravenously, intraarterially, conventionally employed as a solvent or Suspending medium. intramuscularly, Subcutaneously, rectally, vaginally, trans For this purpose any bland fixed oil can be employed includ dermally, intraperitoneally, and intrathecally. In certain ing synthetic mono- or diglycerides. In addition, fatty acids embodiments, the agent is administered parenterally. In other Such as oleic acid are used in the preparation of injectables. embodiments, the agent is administered orally. 0069. In the use of PBA, TUDCA, or TMAO, the agent is 0072 The injectable formulations can be sterilized, for preferably administered orally; however, any of the adminis example, by filtration through a bacteria-retaining filter, or by tration routes listed above may also be used. In certain incorporating sterilizing agents in the form of sterile solid embodiments, the PBA, TUDCA, or TMAO is administered compositions which can be dissolved or dispersed in sterile parenterally. PBA is administered in doses ranging from 10 water or other sterile injectable medium prior to use. mg/kg/day to 5 g/kg/day, preferably from 100 mg/kg/day to 1 0073. The pharmaceutical compositions of the invention g/kg/day, more preferably from 250 mg/kg/day to 750 mg/kg/ may be provided in a kit with other agents used to treat day. TUDCA is administered in doses ranging from 10 diabetes, insulin resistance, or obesity. The kit may include mg/kg/day to 5 g/kg/day, preferably from 100 mg/kg/day to 1 instructions for the treating physician and/or patient, which g/kg/day, more preferably from 250 mg/kg/day to 750 mg/kg/ may include dosing information, safety information, list of day. TMAO is administered in doses ranging from 10 g/kg/ side effects, chemical formula of agent, mechanism of action, day to 0.1 g/kg/day, preferably from 5 g/kg/day to 0.5 g/kg/ etc. In certain embodiments, the kit may include materials for day, more preferably from 2.5 g/kg/day to 500 mg/kg/day. In administering the pharmaceutical composition. For example, certain embodiments, the agent is administered in divided the kit may include a syringe, needle, alcohol Swaps, etc. for doses (e.g., twice per day, three times a day, four times a day, the administration of an injectable preparation. In certain five times a day). In other embodiments, the agent is admin embodiments when two or more agents are provided in a kit, istered in a single dose per day. the active pharmaceutical ingredients may be formulated separately or together. For example, the kit may include a first Pharmaceutical Compositions container with as ER stress modulator (e.g., PBA, TUDCA, 0070 Pharmaceutical compositions of the present inven TMAO, or a derivative thereof) and a second container with a tion and for use in accordance with the present invention may second agent used in treating type 2 diabetes, insulin resis US 2010/0075894 A1 Mar. 25, 2010

tance, hyperglycemia, obesity, or a related disorder (e.g., not experiencing ER stress before they are contacted with the anti-diabetic agents, anti-obesity agents, anti-dyslipidemia test compound. After the cells are contacted with the test agent or anti-atherosclerosis agent, anti-obesity agent, Vita compound, an agent known to cause ER stress is added to the mins, minerals, and anti-hypertensive agents, as described cells, and then the level of at least one ER markers is measured above). In certain embodiments, the active pharmaceutical to determine whether the compound is able to prevent ER ingredients are formulated separately. In other embodiments, stress. As would be appreciated by one of skill in this art, the the active pharmaceutical ingredients are formulated test compound may be tested at various concentrations and together. under various conditions. 0079 Agents identified by the methods of the invention Screening for ER Stress Reducers may be further tested for toxicity, pharmacokinetic proper 0.074 As demonstrated herein, ER stress has been identi ties, use in Vivo, etc. So that they may be formulated and used fied as a target for the treatment of various diseases including in the clinic to treat obesity, type 2 diabetes, hyperglycemia, obesity, type 2 diabetes, insulin resistance, and hyperglyce and insulin resistance. The identified agents may also find use mia. Markers of ER stress have also been identified. With the in the treatment of other diseases associated with ER stress. need for new pharmaceutical agents that reduce or prevent ER Diagnosing Conditions Associated with ER Stress stress, a method of identifying or screening for ER stress 0080. The identification of various ER stress markers modulators is needed. allows for the diagnosis of conditions associated with ER 0075. In certain embodiments, a chemical compound or a stress and the screening of Subjects at risk for developing collection of chemical compounds is assayed to identify com conditions associated with ER stress. Obesity, hyperglyce pounds that reduce or modulate ER stress in vivo or in vitro, mia, type 2 diabetes, and insulin resistance have all been preferably in vivo. These compounds may be any type of shown to be associated with ER stress. Therefore, measuring chemical compound including Small molecules, proteins, the level of an ER stress marker(s) in a subject allows for peptides, polynucleotides, carbohydrates, lipids, etc. In cer determining whether a patient is at risk for any of these tain embodiments, a collection of compounds is screened conditions. Measuring the levels of ER stress markers may be using the inventive method. These collections may be histori used to determine the risk of developing any conditions asso cal libraries of compounds from pharmaceutical companies. ciated with ER stress (e.g., cystic fibrosis, Alzheimer's Dis The collection may also be a combinatorial library of chemi ease). cal compounds. The collection may include at least 5, 10, 50. 0081 ER stress markers that have been identified include 100, 500, 1000, 10000, 100000, or 1000000 compounds. spliced forms of XBP-1, the phosphorylation status of PERK, 0076. The compounds are contacted with cells. The cells the phosphorylation status of eIF2C, mRNA levels of GRP78/ may be any type of cells with an endoplasmic reticulum. The BIP protein levels of GRP78/BIP, and JNK activity. These cells may be animal cells, plant cells, or fungal cells. In ER stress markers may be measured using any techniques certain embodiments, mammalian cells are preferred, par known in the art for measuring mRNA levels, protein levels, ticularly human cells. The cells may be derived from any protein activity, or phosphorylation status. Exemplary tech organ system. In certain embodiments, cells from adipose niques for measuring ER stress markers include western blot tissue or liver tissue are preferred. analysis, northern blot analysis, immunoassays, quantitative 0077. In screening for agents that reduce ER stress, the test PCR analysis, and enzyme activity assay (for a more detailed compound is contacted with a cell already experiencing ER description of these techniques, please see Ausubel et al. stress. The ER stress in the cell may becaused by any tech Current Protocols in Molecular Biology (John Wiley & Sons, niques known in the art. For example, ER stress may be due to Inc., New York, 1999); Molecular Cloning. A Laboratory a genetic alteration in the cells (e.g., XBP-1 mutations) or the Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis treatment with a chemical compound known to cause ER (Cold Spring Harbor Laboratory Press: 1989); each of which stress (e.g., tunicamycin, thapsigargin). The level of ER stress is incorporated herein by reference). markers is assayed before and after addition of the test com I0082 In determining whether a subject is at risk for a pound to determine if the compound reduces ER stress. Mark condition associated with ER stress, one may determine the ers of ER stress that may be assayed in the inventive method levels of one, two, three, four, five, or six ER stress markers. include spliced forms of XBP-1, the phosphorylation status of In certain embodiments, the level of only one ER stress PERK (e.g., Thr980), the phosphorylation status of eIF2C. marker is determined. In other embodiments, the levels of at (e.g., Ser51), mRNA and/or protein levels of GRP78/BIP, and least two ER stress markers are determined. In yet other JNK activity. In certain embodiments, one ER marker is mea embodiments, the levels of at least three ER stress markers are sured. In other embodiments, the levels of a combination of determined. two, three, four, five, six, or more ER stress markers are I0083) Typically, if the level of an ER stress marker is measured. Test compounds that reduce the levels of ER stress determined to be increased as compared to a normal control, markers by at least about 10%, 20%, 30%, 40%, 50%, 60%, the Subject tested is considered at risk for insulin resistance, 70%, 80%, 90%, 95%, 99%, or 100%, preferably at least obesity, hyperglycemia, or type 2 diabetes. The identified 25%, more preferably at least 50%, are considered useful for Subject may then be subject to further testing, treatment may evaluation of ER stress reducers in the clinic. As would be be begun, or the subject may be watched for the future devel appreciated by one of skill in this art, the test compound may opment of symptoms and signs associated with insulin resis be tested at various concentrations and under various condi tance, obesity, hyperglycemia, or type 2 diabetes. The next tions (e.g., various cell types, various causes of ER stress course of action is typically determined by the subject's (genetic vs. chemical), various formulations). health care provider in consultation with the subject. 0078. In another aspect, the invention provides for a I0084. The levels of ER stress markers may be determined method of identifying compounds that prevent ER stress. In for any cells in the subject's body. Preferably, the cells are screening for compounds that prevent ER stress, the cells are connected to the condition being test for. For example, in US 2010/0075894 A1 Mar. 25, 2010

testing for a condition Such as obesity, type 2 diabetes, insulin I0089. We also tested adipose and muscle tissues, impor resistance, or hyperglycemia, hepatocytes or adipocytes may tant sites for metabolic homeostasis, for indications of ER be used. In certain embodiments, hepatocytes are used. In stress in obesity. Similar to liver, PERK phosphorylation, other embodiments, the cells are adipocytes. The cells may be JNK activity, and GRP78 expression were all significantly obtained from the subject by liver biopsy in the case of hepa increased in adipose tissue of obese animals compared with tocytes. Adipocytes may be obtained by biopsy of the subject. lean controls (FIG. 10A-C). However, no indication for ER 0085. The invention also provides kits and systems for stress was evident in the muscle tissue of obese animals (data measuring the levels of various ER stress markers in a subject. not shown). Taken together, these results indicate that obesity The kit may include primers, hybridization probes, poly is associated with induction of ER stress predominantly in nucleotides, antibodies, antibody fragments, gels, buffers, liver and adipose tissues. enzyme substrates, ATP or other nucleotides, tools for obtain ing cells or a biopsy from the Subject, instructions, Software, ER Stress Inhibits Insulin Action in Liver Cells etc. These materials for performing the diagnostic method may be conveniently packaged for use by a physician, Scien (0090. To investigate whether ER stress interferes with tist, pathologist, nurse, lab technician, or health care profes insulin action, we pretreated Fao liver cells with tunicamycin sional. and thapsigargin, agents commonly used to induce ER stress. Tunicamycin significantly decreased insulin-stimulated I0086. These and other aspects of the present invention will tyrosine phosphorylation of IRS-1 (FIGS. 11A and 11B) and be further appreciated upon consideration of the following it also produced an increase in the molecular weight of IRS-1 Examples, which are intended to illustrate certain particular (FIG. 11A). IRS-1 is a substrate for insulin receptor tyrosine embodiments of the invention but are not intended to limit its kinase and serine phosphorylation of IRS-1, particularly Scope, as defined by the claims. mediated by JNK, reduces insulin receptor signaling (Hiro sumi et al., Nature 420:333 (2002); incorporated herein by EXAMPLES reference). Pretreatment of Fao cells with tunicamycin pro Example 1 duced a significant increase in serine phosphorylation of IRS-1 (FIGS. 11A and 11B). Tunicamycin pretreatment also Endoplasmic Reticulum Stress Links Obesity, Insu Suppressed insulin-induced Akt phosphorylation, a more dis lin Action, and Type 2 Diabetes tal event in insulin receptor signaling pathway (FIGS. 11A and 11B). Similar, results were also obtained following treat Results ment with thapsigargin (FIG. 12A), which was independent Induction of ER Stress in Obesity of alterations in cellular calcium levels (FIG. 12B). 0091. We next examined the role of JNK in IRS-1 serine 0087. To examine whether ER stress is increased in obe phosphorylation and inhibition of insulin-stimulated IRS-1 sity, we investigated the expression patterns of several tyrosine phosphorylation by ER stress (FIGS. 11C and 11D). molecular indicators of ER stress in dietary (high fat diet Inhibition of JNK activity with the synthetic inhibitor, induced) and genetic (ob?ob) models of murine obesity. The SP600 125, reversed the ER stress-induced serine phosphory pancreatic ER kinase or PKR like kinase (PERK) is an ER lation of IRS-1 (FIGS. 11C and 11D). Pretreatment of Fao transmembrane protein kinase that phosphorylates the C. Sub cells with a highly specific inhibitory peptide derived from unit of translation initiation factor 2 (eIF2C.) in response to ER the JNK binding protein, JIP (Barr et al., J. Biol. Chem. stress (Shi et al., Mol. Cell. Biol. 18,7499 (1998); Harding et 277:10987 (2002); incorporated herein by reference), also al., Nature 397, 271 (Jan. 21, 1999); each of which is incor completely preserved insulin receptor signaling in cells porated herein by reference). The phosphorylation status of exposed to tunicamycin (FIGS. 11E and 11F). Similar results PERK and elF2C. is therefore a key indicator of the presence were obtained with the synthetic JNK inhibitor, SP600 125 of ER stress. We determined the phosphorylation status of (data not shown). These results indicate that ER stress pro PERK (Thr980) and elF2C. (Ser51) using phospho-specific motes a JNK-dependent serine phosphorylation of IRS-1, antibodies. These experiments demonstrated increased which in turn inhibits insulin receptor signaling. PERK and eIF2C. phosphorylation in liver extracts of obese mice compared with lean controls (FIGS. 1A and 1B). ER stress also leads to JNK activation. Consistent with earlier IRE-1 Plays a Crucial Role in Insulin Receptor Signaling observations (J. Hirosumi et al., Nature 420, 333 (2002); 0092. In the presence of ER stress, increased phosphory incorporated herein by reference), total JNK activity, indi lation of inositol requiring kinase-1C. (IRE-1C) leads to cated by c-Jun phosphorylation, was also dramatically recruitment of TNF-C. receptor-associated factor 2 (TRAF2) elevated in the obese mice (FIGS. 1A and 1B). protein and activates JNK (Urano et al., Science 287:664 0088. The 78 kDa glucose regulated/binding Ig protein (2000); incorporated herein by reference). To address (GRP78/BIP) is an ER chaperone whose expression is whether ER stress-induced insulin resistance is dependent on increased upon ER stress. The GRP78/BIP mRNA levels intact IRE-1C, we measured JNK activation, IRS-1 serine were elevated in the liver tissue of obese mice compared with phosphorylation, and insulin receptor signaling upon expo matched lean controls (FIGS. 1C and 1D). Since GRP78 sure of IRE-1C. and wild type (WT) fibroblasts to tunica expression is responsive to glucose, we tested whether this mycin. In the WT but not IRE-1C. cells, induction of ER upregulation might simply be due to increasing glucose lev stress by tunicamycin resulted in strong activation of JNK els. Treatment of cultured rat Fao liver cells with high levels (FIG. 11G). Tunicamycin also stimulated phosphorylation of of glucose resulted in reduced GRP78 expression (FIG.9A). IRS-1 at Ser307 residue in WT (FIG. 11G) but not IRE-1C. Similarly GRP78 levels were not increased in a mouse model fibroblasts (FIG. 11E). Importantly, tunicamycin inhibited of hyperglycemia (FIG. 9B), indicating that regulation in insulin-stimulated tyrosine phosphorylation of IRS-1 in the obesity is unlikely to be related to glycemia alone. WT cells, whereas no such effect was detected in the IRE US 2010/0075894 A1 Mar. 25, 2010

1C cells (FIG. 11H). The level of insulin-induced tyrosine stimulated tyrosine phosphorylation of the insulin receptor phosphorylation of IRS-1 was dramatically higher in IRE was normal in these cells (FIG. 14). 1C cells despite lower total IRS-1 protein levels (FIG. 11H). These results demonstrate that ER stress-induced inhi XBP-1" Mice Show Impaired Glucose Homeostasis bition of insulin action is mediated by an IRE-1C.-JNK-de pendent protein kinase cascade. (0095 Complete XBP-1 deficiency results in embryonic lethality (Reimold et al., Genes Dev. 14:152, 2000; incorpo Manipulation of XBP-1 Levels Alters Insulin Receptor Sig rated herein by reference). To investigate the role of XBP-1 in naling ER stress, insulin sensitivity and systemic glucose metabo lism in vivo, we studied Balb/C-XBP-1" mice with a null 0093. The transcription factor XBP-1 is a bZIP protein. mutation in one XBP-1 allele. We studied mice on the Balb/C The spliced or processed form of XBP-1 (XBP-1s) is a key genetic background, since this strain exhibits strong resis factor in the transcriptional regulation of molecular chaper tance to obesity-induced alterations in systemic glucose ones and enhances the compensatory UPR (Calfon et al., metabolism. Based on our results with cellular systems, we Nature 415, 92 (2002); Shen et al., Cell 107:893 (2001); hypothesized that XBP-1 deficiency would predispose mice Yoshida et al., Cell 107:881 (2001); Lee et al., Mol. Cell Biol. to the development of insulin resistance and type 2 diabetes. 23:7448 (2003); each of which is incorporated herein by 0096] We placed XBP-1" mice and their WT littermates reference). We therefore reasoned that modulation of XBP-1s on a high fat diet (HFD) at 3 weeks of age. In parallel, control levels in cells should alter insulin action via its potential mice of both genotypes were placed on a chow diet. The total impact on the magnitude of the ER stress responses. To test body weights of both genotypes were similar on chow diet this possibility, we established XBP-1 gain- and loss-of-func and until 12 weeks of age on HFD. After this period, the tion cellular models. First, we established an inducible gene XBP-1" animals on HFD exhibited a small but significant expression system where exogenous XBP-1s is expressed increase in body weight (FIG. 15A). Serum levels of leptin, only in the absence of tetracycline/doxycycline (FIG. 13A). adiponectin and triglycerides did not exhibit any statistically In parallel, we also studied MEFs derived from XBP-1 significant differences between the genotypes measured after mouse embryos (Lee et al., Mol. Cell Biol. 23, 7448 (2003); 16 weeks of HFD (FIG. 16). A. M. Reimold et al., Genes Dev. 14, 152 (2000); Reimold et 0097. On HFD, XBP-1" mice developed continuous and al., Nature 412, 300 (2001); each of which is incorporated progressive hyperinsulinemia evident as early as 4 weeks herein by reference) (FIG. 13B). In fibroblasts without exog (FIG. 15B). Insulin levels continued to increase in XBP-1" enous XBP-1s expression, tunicamycin treatment (2 g/ml) mice for the duration of the experiment. Blood insulin levels resulted in strong PERK phosphorylation starting at 30 min in XBP-1" mice were significantly lowerthan those in XBP utes and peaking at 3-4 hours associated with a mobility shift 1" littermates (FIG. 15B). As shown in FIG. 15C, C-peptide characteristic of PERK phosphorylation (FIG. 13C). In these levels were also significantly higher in XBP-1" animals than cells, there was also a rapid and robust activation of JNK in in WT controls. Blood glucose levels also began to rise in the response to ER stress (FIG. 13C). Upon induction of XBP-1s XBP-1" mice on HFD starting at 8 weeks and remained high expression, there was a dramatic reduction in both PERK until the conclusion of the experiment at 20 weeks (FIG. phosphorylation and JNK activation following tunicamycin 15D). This pattern was the same in both fasted (FIG.15D) and treatment (FIG. 13C). Hence, overexpression of XBP-1s ren fed (data not shown) states. The rise in blood glucose in the dered WT cells refractory to ER stress. Similar experiments face of hyperinsulinemia in the mice on HFD is a strong performed in XBP-1 MEF's revealed an opposite pattern indicator of the development of peripheral insulin resistance. (FIG. 13D). XBP-1 MEF's mounted a strong ER stress 0098. To investigate systemic insulin sensitivity, we per response even when treated with a low dose of tunicamycin formed glucose (GTT) and insulin (ITT) tolerance tests in (0.5 lug/ml), that failed to stimulate significant ER stress in XBP-1" mice and XBP-1" controls. Exposure to HFD WT cells (FIG. 13D). Under these conditions, PERK phos resulted in significant glucose intolerance in XBP-1" mice. phorylation and JNK activation levels in XBP-1, MEFs Upon glucose challenge after 7 weeks of HFD, XBP-1" were significantly higher than those seen in WT controls mice showed significantly higher glucose levels than XBP (FIG. 13D), indicating that XBP-1 cells are prone to ER 1" mice (FIG. 15E). This glucose intolerance continued to stress. Thus alterations in the levels of cellular XBP-1s pro be evident in XBP-1" mice compared with WT mice after 16 tein result in alterations in the ER stress response. weeks on HFD (FIG. 15F). During ITT, the hypoglycemic 0094. Next, we examined whether these differences in the response to insulin was also significantly lower in XBP-1" ER stress responses produced alterations in insulin action as mice compared with XBP-1" littermates at 8 weeks of HFD assessed by IRS-1 serine phosphorylation and insulin-stimu (FIG. 15G) and this reduced responsiveness continued to be lated IRS-1 tyrosine phosphorylation. Tunicamycin-induced evident after 17 weeks of HFD (FIG. 15H). Examination of IRS-1 serine phosphorylation was significantly reduced in islets morphology and function did not reveal significant dif fibroblasts exogenously expressing XBP-1s, compared with ferences between genotypes (FIG. 17). Hence, loss of an that of control cells (FIG. 13E). Upon insulin stimulation, the XBP-1 allele predisposes mice to diet-induced insulin resis extent of IRS-1 tyrosine phosphorylation was significantly tance and diabetes. higher in cells overexpressing XBP-1s, compared with con trols (FIG. 13F). In contrast, IRS-1 serine phosphorylation was strongly induced in XBP-1 MEFs compared with Increased ER Stress and Impaired Insulin Signaling in XBP XBP-1" controls even at low doses of tunicamycin treat 1 */ Mice ment (0.5 lug/ml) (FIG. 13G). Following insulin stimulation, 0099 Our experiments with cultured cells demonstrated the level of IRS-1 tyrosine phosphorylation was significantly an increase in ER stress and a decrease in insulin signaling decreased in tunicamycin-treated XBP-1 cells compared capacity in XBP-1-deficient cells and reversal of these phe with tunicamycin-treated WT controls (FIG. 13H). Insulin notypes upon expression of high levels of XBP-1s. If this US 2010/0075894 A1 Mar. 25, 2010

mechanism is the basis of the insulin resistance seen in XBP Subject to selection. This selection might be a potential under 1" mice, these animals should exhibit high levels of ER lying mechanism for the dramatically high prevalence of stress coupled with impaired insulin receptor signaling. To metabolic diseases in modern times as exposure to excess test this, we first evaluated ER stress by examining PERK caloric load would create continuous stress for ER. In terms phosphorylation and JNK activity in the livers of obese XBP of therapeutics, our findings Suggest that manipulation of the 1" and WT mice. These experiments revealed an increase in ER stress response offers new opportunities for preventing PERK levels and seemingly an increase in liver PERK phos phorylation in obeseXBP-1" mice compared with WT con and treating type 2 diabetes. trols on HFD (FIG. 18A). There was also a significant increase in JNK activity in XBP-1" mice compared with Materials and Methods WT controls (FIG. 18B). Consistent with these results, serine 0103 Biochemical Reagents: Anti-IRS-1, anti-phospho 307 phosphorylation of IRS-1 was also increased in XBP-1" IRS-1 (Ser307) and anti-IRS-2 antibodies were from Upstate mice compared with WT controls on HFD (FIG. 18C). Biotechnology (Charlottesville, Va.). Antibodies against Finally, we studied in vivo insulin-stimulated insulin recep phosphotyrosine, eIF2C, insulin receptor B Subunit, and tor-signaling capacity in these mice. There was no detectable XBP-1 were from Santa Cruz Biotechnology (Santa Cruz, difference in any of the insulin receptor signaling components Calif.). Anti-phospho-PERK, anti-Akt, and anti-phospho in liver and adipose tissues between genotypes on regular diet Akt antibodies and c-Jun protein were from Cell Signaling (FIG. 19). However, following exposure to HFD, major com Technology (Beverly, Mass.). Anti-phospho-elF2C. antibody ponents of insulin receptor signaling in the liver, including was purchased from Stressgen (Victoria, British Columbia, insulin-stimulated IR, IRS-1 and IRS-2 tyrosine- and Akt Canada). Anti-insulin antibody and C-peptide RIA kit were serine-phosphorylation were all decreased in XBP-1" mice purchased from Linco Research (St. Charles, Mo.). Anti compared with WT controls (FIG. 18D-G). A similar sup glucagon antibody was from Zymed (San Francisco, Calif.). pression of insulin receptor signaling was also evident in the PERK antiserum was kindly provided by Dr. David Ron adipose tissues of XBP-1" mice compared with XBP-1" (New York University School of Medicine). Texas red conju mice on HFD (FIG. 20). The suppression of IR tyrosine gated donkey anti-guinea pig IgG and fluorescein-conjugated phosphorylation in XBP-1" mice differs from the observa (FITC-conjugated) goat anti-rabbit IgG were from Jackson tions made in XBP-1 cells where ER stress inhibited insu Immuno Research Laboratories (West Grove, Pa.). Thapsi lin action at a post-receptor level. It is likely that this reflects gargin, tunicamycin, and JNK inhibitors were from Calbio the effects of chronic hyperinsulinemia in vivo on insulin chem (San Diego, Calif.), Insulin, glucose, and Sulindac sul receptors. Hence, our data demonstrate the link between ER fide were from Sigma (St. Louis, Mo.). The Ultra Sensitive stress and insulin action in vivo but are not conclusive in Rat Insulin ELISAkit was from Crystal Chem. Inc. (Downers determining the exact locus in insulin receptor signaling path Grove, Ill.). way that is targeted through this mechanism. 0104 Cells: Rat Fao liver cells were cultured with RPMI 1640 (Gibco, Grand Island, N.Y.) containing 10% fetal Discussion bovine serum (FBS). At 70-80% confluency, cells were serum 0100. In this study, we identify ER stress as a molecular depleted for 12 hours before starting the experiments. link between obesity, the deterioration of insulin action and Reagents including tunicamycin, thapsigargin, and JNK the development of type 2 diabetes. inhibitors were gently added to the culture dishes in the incu 0101 Our findings point to a fundamental mechanism bator to prevent any environmental stress. JNK inhibitors underlying the molecular sensing of obesity-induced meta were added 1 hour before tunicamycin/thapsigargin treat bolic stress by the ER and inhibition of insulin action that ment. The XBP-1 mouse embryonic fibroblasts (MEF) (A. ultimately leads to insulin resistance and type 2 diabetes. We H. Lee, N. N. Iwakoshi, L. H. Glimcher, Mol. Cell. Biol. postulate that ER stress underlies the emergence of the stress 23:7448 (2003); incorporated herein by reference), IRE and inflammatory responses in obesity and the integrated 1C MEF cells (kindly provided by Dr. David Ron, New deterioration of systemic glucose homeostasis (Shi et al., York University School of Medicine), and their wild type Sonenberg, Endocr: Rev. 24:91 (2003); incorporated herein controls were cultured in Dulbecco's Modified Eagle by reference). Our findings differ sharply form earlier work in Medium (DMEM) (Gibco, Grand Island, N.Y.) containing this area linking ER stress to type 1 diabetes and demonstrate 10% FBS. A similar protocol was followed for experiments in that ER stress is an integral mechanism underlying insulin MEF cells, except that the cells were serum depleted for only resistance and type 2 diabetes. 6 hours. 0102 The critical role of ER stress responses in insulin 0105. Overexpression of XBP-1s in MEFs: MEF-tet-off action may represent an evolutionarily conserved mechanism cells (BD Biosciences Clontech, Palo Alto, Calif.) were cul by which stress signals are integrated with metabolic regula tured in DMEM with 100 ug/ml G418 and 1 g/ml doxycy tory pathways. Such integration through ER stress would cline. The MEF-tet-off cells express exogenous tTA (tetracy have been advantageous since proper regulation of energy cline-controlled transactivator) protein, which binds to TRE fluxes and Suppression of major anabolic pathways Such as (tetracycline response element) and activates transcription insulin action might be favorable during acute stress, patho only in the absence oftetracycline or doxycycline. The cDNA gen invasion and immune responses. However, in the pres of the spliced form of XBP-1 was ligated into pTRE2hyg2 ence of a chronic ER stress such as in obesity, this close link plasmid (BD Biosciences Clontech, Palo Alto, Calif.). The between ER stress and metabolic regulation would lead to MEF-tet-off cells were transfected with the TRE2hyg2-XBP development of insulin resistance and eventually, type 2 dia 1s plasmid, followed by selection in the presence of 400 betes. Finally, if the integration of stress signals and meta ug/ml hygromycin B. Individual clones of stable transfectants bolic homeostasis through ER stress has a potential positive were isolated and doxycycline-dependent XBP-1s expression impact on Survival, a highly responsive system would be was confirmed by immunoblotting. US 2010/0075894 A1 Mar. 25, 2010

0106 Northern Blot Analysis: Total RNA was isolated ordinary skill in the art will appreciate that various changes from mouse liver using Trizol reagent (Invitrogen, Carlsbad, and modifications to this description may be made without Calif.), separated by 1% agarose gel, and then transferred departing from the spirit or scope of the present invention, as onto BrightStar Plus nylon membrane (Ambion, Austin, defined in the following claims. Tex.). GRP78 cDNA probe was prepared from mouse liver total cDNAs by RT-PCR using the following primers: 5'-TG APPENDIX A GAGTTCCCCAGATTGAAG-3' and 5'-CCTGACCCAC Drugs for Used in Combination with Er Stress CTTTTTCTCA-3'. The DNA probes were labeled with P Modulators dCTP using random primed DNA labeling kit (Roche, Indianapolis, Ind.). Hybridization was performed according Anti-Diabetic Drugs to the manufacturer's protocol (Ambion, Austin, Tex.) and Peroxisome Proliferator-Activated Receptor-Gamma Ago visualized by Versa Doe Imaging System 3000 (BioRad, Her nists cules, Calif.). 01.07 Protein Extracts From Cells: At the end of each 0111 Rosiglitazone (GlaxoSmithKline's Avandia) treatment, cells were immediately frozen in liquid nitrogen 0112 Pioglitazone (Takeda/Eli Lilly's Actos) and kept at -80°C. Protein extracts were prepared with alysis 0113 Isaglitazone (Mitsubishi Pharma) buffer containing 25 mM Tris-HCl (pH7.4), 2 mM NaVO, 0114 rivoglitazone (Sankyo's CS-011) 10 mM NaF. 10 mM Na P.O., 1 mM EGTA, 1 mM EDTA, 0115 T-131 (Amgen) 1% NP-40, 5 g/ml leupeptin, 5 lug/ml aprotinin, 10 nM 0116 MBX-102 (Metabolex) okadaic acid, and 1 mM phenylmethylsulfonyl fluoride 0117 R-483 (Roche Chugai) (PMSF). Immunoprecipitations and immunoblotting experi 0118 CLX-0921 (Calyx) ments were performed with 750 lug and 75 lug total protein, respectively. Biguanides 0108 Animal Studies and Obesity Models: Adult (10-12 0119 Metformin (Bristol-Myers Squibb’s Glucophage, weeks of age) male ob?ob mice and their wild type (WT) generics, Bristol-Myers Squibb’s Glucophage XR) littermates were purchased from Jackson Labs. Mice used in the diet-induced obesity model were male C57BL/6. All mice Sulfonylureas were placed on high fat diet (HFD: 35.5% fat, 20% protein, 32.7% carbohydrates, Bio-Serve) immediately after weaning 0120 Glimepiride (Aventis’s Amaryl) (at -3 weeks of age). The XBP-1" and XBP-1** mice were 0121 Glyburide/Glibenclamide (Aventis’s Diabeta, on Balb/C genetic background. Insulin and glucose tolerance Pharmacia's Micronase, generics, Pharmacia' s Gly tests were performed as previously described (Hirosumi et al., nase) Nature 420:333 (2002); incorporated herein by reference). 0.122 Glipizide (Pfizer's Glucotrol, generics, Pfizer's Insulin and C-peptide ELISA were performed according to Glucotrol XL) manufacturer's instructions using mouse standards (Crystal (0123 Gliclazide (Servier's Diamicron, Molteni & C.F. Chem. Inc., Downers Grove, Ill.). Pancreas isolated from LLI Alitti's Diabrezide, Irex-Synthélabo's Glycemirex, 16-week-old mice was fixed in Bouin's fluid and formalin, Dainippon's Glimicron, generics) and paraffin sections were double-stained with guinea pig anti-insulin and rabbit anti-glucagon antibodies. Texas red Combination Agents dye conjugated donkey anti-guinea pig IgG and FITC conju (0.124 Metformin/Glyburide (Bristol-Myers Squibb's gated Goat anti-rabbit IgG were used as secondary antibod Glucovance, Hoechst's Suguan M) 1CS 0.125 Metformin/Rosiglitazone (GlaxoSmithKline's 0109 Insulin Infusion and Tissue Protein Extraction: Avandamet) Insulin was injected through the portal vein as previously (0.126 Metformin/Glipizide (Bristol-Myers Squibb's described (Uysal et al., Nature 389:610 (1997); Hirosumi et Metaglip) al., Nature 420:333 (2002); each of which is incorporated herein by reference). Three minutes after insulin infusion, Meglitinides liver was removed and frozen in liquid nitrogen and kept at -80° C. until processing. For protein extraction, liver tissue (O127 Repaglinide (Novo Nordisk’s Prandin/NovoNorm) (-0.3 g) was placed in 10 ml of lysis buffer containing 25 mM I0128 Nateglinide (Novartis's Starlix/Starsis) Tris-HCl (pH7.4), 10 mM NaVO 100 mM NaF, 50 mM Alpha-Glucosidase Inhibitors NaP.O. 10 mM EGTA, 10 mM EDTA, 1% NP-40, 5ug/ml leupeptin, 5 g/ml aprotinin, 10 nMokadaic acid, and 2 mM I0129 Acarbose (Bayer's Precose/Glucobay, generics) PMSF. After homogenization on ice, the tissue lysate was I0130 Miglitol (Pharmacia's Glyset) centrifuged at 4,000 rpm for 15 minutes at 4°C. followed by I0131 Voglibose (Takeda’s Basen) 55,000 rpm for 1 hour at 4°C. One milligram of total tissue protein was used for immunoprecipitation and Subsequent Insulin and Insulin Analogues immunoblotting, whereas 100-150 ug total tissue protein was 0.132. Insulin lispro (Eli Lilly's Humalog) used for direct immunoblotting (Hirosumi et al., Nature 420: 0.133 Insulin glargine (Aventis’s Lantus) 333 (2002); incorporated herein by reference). I0134) Exubera (Nektar/Pfizer/Aventis) 0.135 AERX Insulin Diabetes Management System (Ara Other Embodiments digm/Novo Nordisk) 0110. The foregoing has been a description of certain non (0.136 AIR inhaled insulin (Eli Lilly/Alkermes) limiting preferred embodiments of the invention. Those of I0137 Oralin (Generex) US 2010/0075894 A1 Mar. 25, 2010 16

0.138. Insulin detemir (NN-304) (Novo Nordisk) Avandamet Tablets (GlaxoSmithKline) 0139 Insulin glulisine (Aventis) 0173 Metformin Hydrochloride, Rosiglitazone Maleate Dual PPAR Agonists and PPAR Pan Agonists Avandia Tablets (GlaxoSmithKline) 0140 BMS-298585 (Bristol-Myers Squibb/Merck) 0.174 Rosiglitazone Maleate 0141 Tesaglitazar (AstraZeneca's Galida) 0142 muraglitazar (BMS-Merck and Co) DiaBeta Tablets (Sanofi-Aventis) 0143 naveglitazar (Lilly-Ligand’s LY-818) 0144) TAK-559 (Takeda) (0175 Glyburide 0145 netoglitazone (Mitsubishi) Fortamet Extended-Release Tablets (Andrx Labs) 0146 GW-677594 (GSK) 0147 AVE-0847 (Aventis) (0176 Metformin Hydrochloride 0148 LY-929 (Lilly-Ligand) 0149 ONO-5129 (ONO) Glucotrol XL Extended Release Tablets (Pfizer) (0177 Glipizide Glucagon-Like Peptide-1 (GLP-1) Analogues and Agonists 0150 Exenatide (AC-2993) (Eli Lilly/Amylin Pharma Metaglip Tablets (Bristol-Myers Squibb) ceuticals) (0178 Glipizide, Metformin Hydrochloride 0151 Exenatide LAR (AC-2993 LAR) (Amylin Pharma ceuticals/Alkermes/Eli Lilly) Prandin Tablets (0.5, 1, and 2 mg) (Novo Nordisk) 0152 Liraglutide (insulinotropin/NN-2211) (Scios/Novo (0179 Repaglinide Nordisk) 0153. CJC-1131 (ConjuChem) Precose Tablets (Bayer) 0154 AVE-0010 (Aventis-Zealand) (O155 BIM-51077 (Roche-Teijin-Ipsen) 0180 Acarbose 0156 NN-2501 (Novo Nordisk) (O157 SUN-E7001 (Daiichi Suntory-Sankyo) Starlix Tablets (Novartis) 0181 Nateglinide Dipeptidyl Peptidase IV (DPP-IV). Inhibitors Other Drugs from the PDR: 0158 LAF-237 (Novartis) Indication=Insulin Dependent Diabetes 0159 MK-431 (Merck and Co) (0160 PSN-9301 (Probiodrug Prosidion) Humalog-Pen (Lilly) (0161 815541 (GlaxoSmithKline-Tanabe) 0182 Insulin Lispro, Human (0162 823093 (GlaxoSmithKline) (0163 825964 (GlaxoSmithKline) Humalog Mix 75/25-Pen (Lilly) (0164 BMS-477118 (BMS) 0183 Insulin Lispro Protamine, Human, Insulin Lispro, Pancreatic Lipase Inhibitors Human (0165 Orlistat (Roche Holding) Humulin 50/50, 100 Units (Lilly) Sodium Glucose co Transporter (SGLT) Inhibitors 0.184 Insulin, Human Regular and Human NPH Mixture (0166 T-1095 (Tanabe-J&J) Humulin 70/30 Pen (Lilly) (0167 AVE-2268 (Aventis) 0185. Insulin, Human Regular and Human NPH Mixture (0168 869682 (GlaxoSmithKline-Kissei) Amylin Analog Humulin L, 100 Units (Lilly) 0169 pramlintide (Amylin's Symlin) 0186 Insulin, Human, Zinc Suspension Other Drugs from the PDR: Humulin N, 100 Units (Lilly) Indication Hyperglycemia 0187. Insulin, Human NPH Actos Tablets (Takeda) Humulin R (U-500) (Lilly) (0170 Pioglitazone Hydrochloride 0188 Insulin, Human Regular Amaryl Tablets (Sanofi-Aventis) Humulin R, 100 Units (Lilly) (0171 Glimepiride 0189 Insulin, Human Regular Apidra Injection (Sanofi-Aventis) Humulin U, 100 Units (Lilly) 0172 Insulin Glulisine 0190. Insulin, Human, Zinc Suspension US 2010/0075894 A1 Mar. 25, 2010

Humulin N Pen (Lilly) Lantus Injection (Sanofi-Aventis) (0191 Insulin, Human NPH 0209 Insulin glargine Innovo (Novo Nordisk) Metaglip Tablets (Bristol-Myers Squibb) (0192 Device 0210 Glipizide, Metformin Hydrochloride Lantus Injection (Sanofi-Aventis) Prandin Tablets (0.5, 1, and 2 mg) (Novo Nordisk) 0193 Insulin glargine 0211 Repaglinide Novolin 70/30 Human Insulin 10 ml Vials (Novo Nordisk) Precose Tablets (Bayer) 0194 Insulin, Human Regular and Human NPH Mixture 0212 Acarbose Novolin 70/30 PenFill 3 ml Cartridges (Novo Nordisk) Starlix Tablets (Novartis) (0195 Insulin, Human Regular and Human NPH Mixture 0213 Nateglinide Other Drugs from the PDR: Novolin N Human Insulin 10 ml Vials (Novo Nordisk) 0196. Insulin, Human NPH Indication=Type 1 Diabetes Lantus Injection (Sanofi-Aventis) Novolin N PenFill 3 ml Cartridges (Novo Nordisk) 0214 Insulin glargine 0.197 Insulin, Human NPH Novolin 70/30 InnoLet (Novo Nordisk) Novolin R Human Insulin 10 ml Vials (Novo Nordisk) 0215. Insulin, Human Regular and Human NPH Mixture 0198 Insulin, Human Regular Novolin N InnoLet (Novo Nordisk) Novolin R PenFill 1.5 ml Cartridges (Novo Nordisk) 0216. Insulin, Human NPH (0199 Insulin, Human Regular Novolin RInnoLet (Novo Nordisk) Novolin R PenFill 3 ml Cartridges (Novo Nordisk) 0217. Insulin, Human NPH 0200 Insulin, Human Regular Other Drugs from the PDR: NovoLog Injection (Novo Nordisk) Indication=Type 2 Diabetes 0201 Insulin Aspart, Human Regular Fortamet Extended-Release Tablets (Andrx Labs) NovoLog Mix 70/30 (Novo Nordisk) 0218 Metformin Hydrochloride 0202 Insulin Aspart Protamine, Human, Insulin Aspart, Lantus Injection (Sanofi-Aventis) Human 0219. Insulin glargine Other Drugs from the PDR: Prandin Tablets (0.5, 1, and 2 mg) (Novo Nordisk) Indication=Non-Insulin Dependent Diabetes 0220 Repaglinide Actos Tablets (Takeda) anti-obesity drugs 0203 Pioglitazone Hydrochloride Pancreatic Lipase Inhibitors Amaryl Tablets (Sanofi-Aventis) 0221) Orlistat (Roche's Xenical, Roche Nippon's Xenical) 0204 Glimepiride Serotonin and Norepinephrine Reuptake Inhibitors 0222 Sibutramine (Abbott/Knoll's Meridia, AstraZen Avandamet Tablets (GlaxoSmithKline) eca's Reductil, Eisai's Reductil) 0205 Metformin Hydrochloride, Rosiglitazone Maleate Noradrenergic Anorectic Agents Avandia Tablets (GlaxoSmithKline) 0223 Phentermine (GlaxoSmithKline's Fastin, Medeva's 0206 Rosiglitazone Maleate Ionamin) 0224 Mazindol (Wyeth-Ayerst's Mazanor, Novartis's Diabeta Tablets (Sanofi-Aventis) Sanorex) 0207 Glyburide Peripherally Acting Agents 0225. ATL-962 (Alizyme) Glucotrol XL Extended Release Tablets (Pfizer) 0226 HMR-1426 (Aventis) 0208 Glipizide 0227 GI-181771 (GlaxoSmithKline) US 2010/0075894 A1 Mar. 25, 2010

Centrally Acting Agents Peptide YY 3-36 0228 Recombinant human ciliary neurotrophic factor 0255 AC162352 (Amylin) (AXokine) (Regeneron) Other Drugs from the PPM: 0229 Rimonabant (SR-141716) (Sanofi-Synthélabo) 0230 BVT-933 (GlaxoSmithKline/BioVitrum) Indication=Obesity 0231 Bupropion SR (GlaxoSmithKline) Adipex-P Capsules (Gate) 0232 P-57 (Phytopharm) (0256 Phentermine Hydrochloride Thermogenic Agents 0233 TAK-677 (AJ-9677) (Dainippon/Takeda) Adipex-P Tablets (Gate) (0257 Phentermine Hydrochloride Cannabinoid CB1 Antagonist Desoxyn Tablets, USP (Ovation) 0234 Acomplia (Sanofi-Aventis) 0235 SLV319 (Solvay) (0258 Methamphetamine Hydrochloride Ciliary Neurotrophic Factor (CNTF) Agonists Ionamin Capsules (Celltech) 0236. Axokine (Regeneron) 0259 Phentermine Resin Other Anti-Obesity Drugs Meridia Capsules (Abbott) 0237) 1426 (Sanofi-Aventis) 0260 Sibutramine Hydrochloride Monohydrate 0238 1954 (Sanofi-Aventis) 0239 c-2624 (Merck & Co) Xenical Capsules (Roche Laboratories) 0240 c-5093 (Merck & Co) 0261 Orlistat 0241 T71 (Tularik) Other Drugs from the PDR: Cholecystokinin (CCK) Agonist Prescribing Category Appetite Suppressant 0242 GI 181771 (GSK) Adipex-P Capsules (Gate) Lipid Metabolism Modulator 0262 Phentermine Hydrochloride 0243 AOD9604 (Monash University/Metabolic Pharma Adipex-P Tablets (Gate) ceuticals) 0263 Phentermine Hydrochloride Lipase Inhibitor Desoxyn Tablets, USP (Ovation) 0244 ATL962 (Alizyme, Takeda) 0264 Methamphetamine Hydrochloride Glucagon-Like Peptide 1 Agonist Ionamin Capsules (Celltech) 0245 AC 137 (Amylin) 0265 Phentermine Resin Leptin Agonist Meridia Capsules (Abbott) 0246 Second generation leptin (Amgen) 0266 Sibutramine Hydrochloride Monohydrate Beta-3 Adrenergic Agonist Anti-Atherosclerosis Drugs 0247 SR58611 (Sanofi-Aventis) 0248 CP33 1684 (Pfizer) HMG-CoA Reductase Inhibitors (Statins) 0249 LY377604 (Eli Lilly) 0267 Atorvastatin (Warner-Lambert/Pfizer's Lipitor) (0250 n5984 (Nisshin Kyorin Pharmaceutical) 0268 Pravastatin (Bristol-Myers Squibb's Pravachol/ Sankyo's Mevalotin) Peptide Hormone 0269. Simvastatin (Merck & Co.'s Zocor) 0251 peptide YY 3-36 (Nastech) (0270. Lovastatin (Merck & Co.'s Mevacor) (0271 Fluvastatin (Novartis's Lescol) CNS Modulator 0272. Cerivastatina (Bayer's Lipobay/GlaxoSmithKline's Baycol) 0252 s2367 (Shionogi & Co Ltd) 0273 Rosuvastatin (AstraZeneca's Crestor) 0274 Pitivastatin (itavastatin/risivastatin) (Nissan/ Neutrotrophic Factor Kowa/Sankyo/Novartis) 0253 Peg axokine (Regeneron) Fibrates 5HT2C Serotonin Receptor Agonist (0275 Bezafibrate (Boehringer Mannheim/Roche's Beza 0254 APD356 (Arena Pharmaceutical) lip, Kissei’s Bezatol) US 2010/0075894 A1 Mar. 25, 2010

0276 Clofibrate (Wyeth-Ayerst's Atromid-S, generics) Bile Acid Modulators (0277 Fenofibrate (Fournier's Lipidil, Abbott’s Tricor, 0303 GT102-279 (GelTex/Sankyo) Takeda’s Lipantil, generics) (0304 HBS-107 (Hisamitsu/Banyu) (0278 Gemfibrozil (Pfizer's Lopid, generics) Peroxisome Proliferation Activated Receptor (PPAR) Ago Bile Acid Sequestrants nists (0279 Cholestyramine Bristol-Myers Squibb's Questran 0305 Tesaglitazar (AZ-242) (AstraZeneca) and Questran Light, generics (0306 Netoglitazone (MCC-555) (Mitsubishi/Johnson & 0280 Colestipol Pharmacia's Colestid Johnson) (0307 GW-409544 (Ligand Pharmaceuticals/Glaxo Niacin SmithKline) 0281 Niacin immediate release (Aventis's Nicobid, (0308 GW-501516 (Ligand Pharmaceuticals/Glaxo Upsher-Smith's Niacor, Aventis's Nicolar, Sanwaka SmithKline) gaku's Perycit, generics 0282 Niacin—extended release (Kos Pharmaceuticals Gene-Based Therapies Niaspan, Upsher-Smith's Slo-Niacin) 0309 AdGVVEGF121.10 (GenVec) 0310 ApoA1 (UCS Pharma/Groupe Fournier) Antiplatelet Agents 0311 EG-004 (Trinam) (Ark Therapeutics) 0283 Aspirin (Bayer's Aspirin, generics) 0312 ATP-binding cassette transporter-A1 (ABCA1) 0284 Clopidogrel (Sanofi-Synthélabo/Bristol-Myers (CV Therapeutics/Incyte, Aventis, Xenon) Squibb's Plavix) 0285) Ticlopidine (Sanofi-Synthélabo's Ticlid, Daiichi's Composite Vascular Protectants Panaldine, generics) 0313 AGI-1067 (Atherogenics) Angiotensin-Converting Enzyme Inhibitors Other Anti-Atherosclerotic Agents 0286 Ramipril (Aventis's Altace) 0314 BO-653 (Chugai Pharmaceuticals) 0287 Enalapril (Merck & Co.'s Vasotec) Glycoprotein IIb/IIIa Inhibitors Angiotensin II Receptor Antagonists 0315 Roxifiban (Bristol-Myers Squibb) 0288 Losartan potassium (Merck & Co.'s Cozaar) 0316 Gantofiban (Yamanouchi) 0317 Cromafiban (Millennium Pharmaceuticals) Acyl CoA Cholesterol Acetyltransferase (ACAT) Inhibitors Aspirin and Aspirin-Like Compounds 0289 Avasimibe (Pfizer) 0290 Eflucimibe (BioMérieux Pierre Fabre/Eli Lilly) 0318 Asacard (slow-release aspirin) (Pharmacia) 0291 CS-505 (Sankyo and Kyoto) 0319 Pamicogrel (Kanebo/Angelini Ricerche/CEPA) 0292 SMP-797 (Sumito) Combination Therapies Cholesterol Absorption Inhibitors 0320 Advicor (niacin/lovastatin) (Kos Pharmaceuticals) 0293 EZetimibe (Schering-Plough/Merck & Co.) 0321 Amlodipine/atorvastatin (Pfizer) Pamaqueside (Pfizer) 0322 Simvastatin/ezetimibe (Merck & Co./Schering 0294 Plough)

Cholesterol Ester Transfer Protein (CETP) Inhibitors IBAT Inhibitors 0295 CP-529414 (Pfizer) S-8921 (Shionogi) 0296 JTT-705 (Japan Tobacco) 0323 0297 CETi-1 (Avant Immunotherapeutics) Squalene Synthase Inhibitors Microsomal Triglyceride Transfer Protein (MTTP) Inhibitors 0324 BMS-188494. I(Bristol-Myers Squibb) 0325 CP-210172 (Pfizer) 0298 Implitapide (Bayer) 0326 CP-295697 (Pfizer) 0299 R-103757 (Janssen) 0327 CP-294.838 (Pfizer) Other Cholesterol Modulators Monocyte Chemoattractant Protein (MCP)-1 Inhibitors 0300 NO-1886 (Otsuka/TAP Pharmaceutical) 0328 RS-504393 (Roche Bioscience) 0301 CI-1027 (Pfizer) 0329. Other MCP-1 inhibitors (GlaxoSmithKline, Teijin, 0302) WAY-135433 (Wyeth-Ayerst) and Bristol-Myers Squibb) US 2010/0075894 A1 Mar. 25, 2010 20

Other Drugs from the PDR: Vytorin 10/40 Tablets (Merck/Schering Plough) Indication=Hypercholesterolemia 0348 EZetimibe, Simvastatin Advicor Tablets (Kos) Vytorin 10/40 Tablets (Schering) 0330 Lovastatin, Niacin 0349 EZetimibe, Simvastatin Altoprev Extended-Release Tablets (Andrx Labs) Vytorin 10/80 Tablets (Merck/Schering Plough) 0331 Lovastatin 0350 EZetimibe, Simvastatin Caduet Tablets (Pfizer) Vytorin 10/80 Tablets (Schering) 0332 Amlodipine Besylate, Atorvastatin Calcium 0351 EZetimibe, Simvastatin Crestor Tablets (AstraZeneca) WelChol Tablets (Sankyo) 0333 Rosuvastatin Calcium 0352 Colesevelam Hydrochloride Lescol Capsules (Novartis) Zetia Tablets (Schering) Fluvastatin Sodium 0334 0353 Ezetimibe Lescol Capsules (Reliant) Zetia Tablets (Merck/Schering Plough) 0335) Fluvastatin Sodium 0354 EZetimibe Lescol XL Tablets (Novartis) Zocor Tablets (Merck) 0336 Fluvastatin Sodium 0355 Simvastatin Lescol XL Tablets (Reliant) Anti-Dyslipidemia Drugs 0337 Fluvastatin Sodium HMG-CoA Reductase Inhibitors Lipitor Tablets (Parke-Davis) 0356. Altorvastatin (Pfizer's Lipitor/Tahor/Sortis/Tor 0338 Atorvastatin Calcium vast/Cardyl) 0357 Simvastatin (Merck's Zocor/Sinvacor, Boe Lofibra Capsules (Gate) hiringer Ingelheim's Denan, Banyu's Lipovas) 0358 Pravastatin (Bristol-Myers Squibb's Pravachol, 0339 Fenofibrate Sankyo's Mevalotin/Sanaprav) 0359 Fluvastatin (Novartis's Lescol/Locol/Lochol, Mevacor Tablets (Merck) Fujisawa's Cranoc, Solvay’s Digaril) 0340 Lovastatin 0360 Lovastatin (Merck's Mevacor/Mevinacor, Bex al’s Lovastatina, Cepa; Schwarz Pharma's Liposcler) Niaspan Extended-Release Tablets (Kos) 0361 Rosuvastatin (AstraZeneca's Crestar) 0341 Niacin 0362 Pitavastatin (Nissan Chemical, Kowa Kogyo, Sankyo, and Novartis) Pravachol Tablets (Bristol-Myers Squibb) HMG-CoA Reductase Inhibitor Combination Therapies 0342 Pravastatin Sodium 0363 Simvastatin/ezetimibe (Merck and Schering Tricor Tablets (Abbott) Plough) 0343 Fenofibrate Fibrates Vytorin 10/10 Tablets (Merck/Schering Plough) 0364 Fenofibrate (Abbott's Tricor, Fournier's Lipidil/ Lipantil) 0344 EZetimibe, Simvastatin 0365 Bezafibrate (Roche's Béfizal/Cedur/Bezalip, Kissei’s BeZatol, generics) Vytorin 10/10 Tablets (Schering) 0366 Gemfibrozil (Pfizer's Lopid/Lipur, generics) (0345 EZetimibe, Simvastatin 0367 Clofibrate (Wyeth's Atromid-S, generics) 0368 Ciprofibrate (Sanofi-Synthélabo's Modalim) Vytorin 10/20 Tablets (Merck/Schering Plough) 0346) EZetimibe, Simvastatin Bile Acid Sequestrants 0369 Colestyramine (Bristol-Myers Squibb's Questran) Vytorin 10/20 Tablets (Schering) 0370 Colestipol (Pfizer's Colestid) 0347 EZetimibe, Simvastatin 0371 Colesevelam (Genzyme/Sankyo's Welchol) US 2010/0075894 A1 Mar. 25, 2010 21

Cholesterol Absorption Inhibitors Caduet Tablets (Pfizer) 0372 Ezetimibe (Merck and Schering-Plough's Zetia) 0402 Amlodipine Besylate, Atorvastatin Calcium 0373 Pamaqueside (Pfizer) Crestor Tablets (AstraZeneca) Nicotinic Acid Derivatives 0403 Rosuvastatin Calcium 0374 Nicotinic acid (Kos's Niaspan, Yamanouchi’s Nyclin) Lescol Capsules (Novartis) Acyl-CoA Cholesterol Acyltransferase Inhibitors- 0 0404 Fluvastatin Sodium 0375. Avasimibe (Pfizer) Lescol Capsules (Reliant) 0376 Eflucimibe (Eli Lilly) 04.05 Fluvastatin Sodium Cholesteryl Ester Transfer Protein Inhibitors Lescol XL Tablets (Novartis) 0377 Torcetrapib (Pfizer) 0406 Fluvastatin Sodium 0378 JTT-705 (Japan Tobacco) 0379 CETi-1 (Avant Immunotherapeutics) Lescol XL Tablets (Reliant) Microsomal Triglyceride Transfer Protein Inhibitors- 0 0407. Fluvastatin Sodium 0380 Implitapide (Bayer) Lipitor Tablets (Parke-Davis) 0381 CP-346086 (Pfizer) 0408 Atorvastatin Calcium Peroxisome Proliferation Activated Receptor Agonists Lofibra Capsules (Gate) 0382 GW-501516 (Ligand Pharmaceuticals and Glaxo- 04.09 Fenofibrate SmithKline) 0383 Tesaglitazar (AstraZeneca) Mevacor Tablets (Merck) 0384 LY-929 (Ligand Pharmaceuticals and Eli Lilly) 0410 Lovastatin 0385 LY-465608 (Ligand Pharmaceuticals and Eli Lilly) 0386 LY-518674(Ligand Pharmaceuticals and Eli Lilly) Niaspan Extended-Release Tablets (Kos) (0387 MK-767 (Merck and Kyorin) 0411 Niacin Squalene Synthase Inhibitors Pravachol Tablets (Bristol-Myers Squibb) 0388 TAK-475 (Takeda) 0412 Pravastatin Sodium Other New Approaches Tricor Tablets (Abbott) 0389 MBX-102 (Metabolex) 0390 NO-1886 (Otsuka) 0413 Fenofibrate 0391) Gemeabene (Pfizer) Vytorin 10/10 Tablets (Merck/Schering Plough) Liver X Receptor Agonists 0414 Ezetimibe, Simvastatin 0392 GW-3965 (GlaxoSmithKline) Vytorin 10/10 Tablets (Schering) 0393 TU-0901317 (Tularik) 0415 EZetimibe, Simvastatin Bile Acid Modulators Vytorin 10/20 Tablets (Merck/Schering Plough) 0394 BTG-511 (British Technology Group) 0395 HBS-107 (Hisamitsu and Banyu) 0416 EZetimibe, Simvastatin 0396 BARI-1453 (Aventis) 0397 S-8921 (Shionogi) Vytorin 10/20 Tablets (Schering) 0398 SD-5613 (Pfizer) 0417 EZetimibe, Simvastatin 0399 AZD-7806 (AstraZeneca) Other Drugs from the PDR: Vytorin 10/40 Tablets (Merck/Schering Plough) Indication=Hypercholesterolemia 0418 EZetimibe, Simvastatin Advicor Tablets (Kos) Vytorin 10/40 Tablets (Schering) 0400 Lovastatin, Niacin 0419 EZetimibe, Simvastatin Altoprev Extended-Release Tablets (Andrx Labs) Vytorin 10/80 Tablets (Merck/Schering Plough) 0401 Lovastatin 0420 EZetimibe, Simvastatin US 2010/0075894 A1 Mar. 25, 2010 22

Vytorin 10/80 Tablets (Schering) 0446 Nadolol (Bristol-Myers Squibb/Sanofi-Synthéla bo's Corgard, generics) 0421 EZetimibe, Simvastatin 0447 Penbutolol sulfate (Schwarz Pharma's Levatol, Wolff Aventis's BetaPressin) WelChol Tablets (Sankyo) 0448 Pindolol (Novartis's Visken, generics) 0422 Colesevelam Hydrochloride 0449 Propranolol hydrochloride (AstraZeneca's Inderal/Dociton, Wyeth’s Inderal LA, generics) Zetia Tablets (Schering) 0450 Timolol maleate (Merck & Co.'s Blocadren, generics) 0423 Ezetimibe 0451 Carvedilol (Roche's Coreg/Dilatrend/Coropres/ Zetia Tablets (Merck/Schering Plough) Eucardic, GlaxoSmithKline's Coreg) 0424 EZetimibe Calcium-Channel Blockers 0452 Amlodipine besylate (Pfizer's Amlor/Istin/Nor Zocor Tablets (Merck) vasc) 0425 Simvastatin 0453 Felodipine (AstraZeneca's Flodil/Modip/ Plendil/Splendil) Anti-Hypertension Drugs 0454 Isradipine (Novartis's Lomir/Prescal) 0455 Nicardipine (Roche/Yamanouchi’s Cardene, Diuretics generics) 0426 Chlorthalidone (Alliance's Hygotron, generics) 0456 Nifedipine (Bayer's Adalat, generics) 0427 Metolazone (Generics) 0457 Nisoldipine (AstraZeneca's Sular, Bayer's Sys 0428 Indapamide (Servier's Natrilix/Tertensif, Aven cor MR/Bay mycard) tis's LoZol, Merck’s Indapamide, generics) 0458 Diltiazem hydrochloride (Sanofi-Synthélabo's 0429 Bumetanide (Leo's Burinex, Sankyo's Lune Tildiem Angizem 60, Pfizer's Dinisor, Generics) toron, Roche’s Bumex, generics) 0459 Verapamil Hydrochloride (Knoll's Isoptin Press/ Manidon RetardlSecuron, Pharmacia's Calan/Covera, 0430 Ethacrynic acid (Merck & Co.'s Edecrin) generics) 0431 Furosemide (Aventis's Lasilix/Lasix/Eutensin/ Seguril, generics) 0460 AZelnidipine (Sankyo/UBE) 0461 Pranidipine (Otsuka) 0432 Torsemide (Roche’s Demadex, generics) 0462 Graded diltiazem formulation (BioVail) 0433 Amiloride hydrochloride (Merck & Co.'s 0463 (S)-amlodipine (Sepracor? Emcure) Midamor, generics) 0464 clevidipine (AstraZeneca/The Medicines Com 0434 Spironolactone (Pharmacia's Spirolang/Aldac pany) tone, Mylan's Spironolactone, generics) 0435 Triamterene (GlaxoSmithKline's Dyrenium, Angiotensin-Converting Enzyme Inhibitors Goldshield's Dytiac, Isei’s Triamterene?Triteren) 0465 Benazepril hydrochloride (Novartis’s Cibacen/ Alpha Blockers Lotensin) 0466 Captopril (Bristol-Myers Squibb's Lopril/Lopi 0436 Doxazosin mesylate (Pfizer's Zoxan/Cardura/ rin/Capoten/Acepress, Sanofi-Synthélabo's Alopresin, Cardenalin, Hexyl's Doxacor, generics) generics) 0437. Prazosin hydrochloride (Pfizer's Minipress, 0467 Enalapril maleate (Merck & Co.'s Vasotec, generics) Banyu's Renivoce, generics) 0438 Terazosin hydrochloride (Abbott's Hytrin/Flot 0468 Fosinopril sodium (Bristol-Myers Squibb's Fosi rin/Heitrin/Itrin/Deflox, Mitsubishi Wellpharma's norm/Tensogard/Fosinil/Staril/Monopril) Vasomet, generics) 0469 Lisinopril (Merck & Co.'s Prinivil, AstraZeneca's Zestril) Beta Blockers 0470 Moexipril (Schwarz's Moex/Fempress/Univasc) 0439 Acebutolol (Bayer's Prent, Wyeth's Sectral, 0471 Perindopril (Servier's Conversyl) Aventis’s Acetanol, generics) 0472 Quinapril Hydrochloride (Pfizer's Accupril/ 0440 Atenolol (AstraZeneca/Sumitomo's Tenormin, Acuitel/Accuprin/Acuprel, Sanofi-Synthélabo's Korec, generics) generics) 0441 Betaxolol (Sanofi-Synthélabo's Kerlone, gener 0473 Ramipril (Aventis’s Altace) ics) 0474 Trandolapril (Abbott's Gopten/Mavik) 0442 Bisoprolol fumarate (Merck KGaA's Cardicor, Lipha Sante's Detensiel/Cardensiel, Lederle's Mono Angiotensin II Receptor Antagonists cor/Zebeta, generics) 0475 Losartan (Merck & Co.'s Cozaar) 0443 Carteolol Hydrochloride (Lipha Sante/Otsuka's 0476 Valsartan (Novartis's Tareg/Diovan, Aventis’s Mikelan, Abbott's Cartrol) Nisis) 0444 Metoprolol tartrate (Novartis's Lopressor/Prelis, 0477 Irbesartan (Bristol-Myers Squibb/Sanofi-Synthéla AstraZeneca's Seloken/Betaloc, generics) bo's Aprovel/Karvea) 0445 Metoprolol succinate (AstraZeneca's Toprol-XL 0478 Candesartan (AstraZeneca's Atacand/Ratacand/Ai Herz-mite, generics) nias, Takeda’s Blopress) US 2010/0075894 A1 Mar. 25, 2010 23

0479. Telmisartan (Boehringer Ingelheim's Micardis) 0512 SPP600 (Speedel) 0480 Eprosartan (Solvay’s Teveten) 0513 SPP 800 (Locus/Speedel) 048.1 Olmesartan (Sankyo/Recordati/Menarini/Forest/ Kowa) Angiotensin Vaccines 0482 YM-358 (Yamanouchi) 0514 PMD-3 117 (Protherics) Combination Therapies ACEANEP Inhibitors 0483 Benazepril hydrochloride/hydrochlorothiazide (Novartis's Cibadrex/Lotensin HCT) 0515 AVE-7688 (Aventis) 0484 Captopril/hydrochlorothiazide (Bristol-Myers 0516 GW-660511 (Zambon SpA) Squibb's Capozide/Ecazide) Na+/K+ ATPase Modulators 0485 Enalapril maleate/hydrochlorothiazide (Merck & Co.'s Vasorectic/Co Renitec/Innozide, AstraZeneca's 0517 PST-2238 (Prassis-Sigma-Tau) LeXXel, generics) 0486 Lisinopril/Hydrochlorothiazide (Merck & Co.'s Endothelin Antagonists Prinzide, AstraZeneca’s Zestorectic) 0518 PD-156707 (Pfizer) 0487 Losartan/hydrochlorothiazide (Merck & Co.'s Hyzaar) Vasodilators 0488 Atenolol/Chlorthalidone (AstraZeneca's Teno retic, generics) 0519 NCX-4016 (NicOx) 0489 Bisoprolol fumarate/Hydrochlorothiazide (Led 0520 LP-805 (Pola/Wyeth) erle's Ziac, generics) 0490 Metoprolol tartrate/hydrochlorothiazide (Novar Naturetic Peptides tis's Lopressor HCT, Pharmacia's Selopresin/Selozide) 0521 BDNP (Mayo Foundation) 0491 Amlodipine besylate/benazepril hydrochloride (Pfizer's Norvase, Novartis's Lotrel) Angiotensin Receptor Blockers (ARBs) 0492. Felodipine?enalapril maleate (AstraZeneca's pratosartan (Pratosartan/Boryung) Lexxel) 0522 0493 Verapamil hydrochloride/trandolapril (Knoll/ AGE Crosslink Breakers Abbott's Tarka, Aventis's Udramil) 0494 Lercanidipine and enalapril (Recordati/Pierre 0523 alagebrium chloride (Alteon) Fabre) 0495 Olmesartan/hydrochlorothiazide (Sankyo) Endothelin Receptor Antagonists 0496 Eprosartan/hydrochlorothiazide (Unimed) 0524 Tezosentan (Genentech) 0497 Amlodipine besylate/atorvastatin (Pfizer) 0525 Ambrisentan (Myogen) 0498 Nitrendipine/enalapril (Vita Invest) 0526 BMS193884 (BMS) Vasopeptidase Inhibitors 0527 Sitaxsentan (Encysive Pharmaceuticals) 0528 SPP301 (Roche/Speedel) 0499. Omapatrilat (Bristol-Myers Squibb) 0529) Darusentan (Myogen/Abbott) (0500 Gemopatrilat (Bristol-Myers Squibb) 0530 J104132 (Banyu/Merck & Co.) 0501) Fasidotril (Eli Lilly) 0531 TBC3711 (Encysive Pharmaceuticals) 0502 sampatrilat (Pfizer/Shire) 0532 SB 234551 (GSK/Shionogi) 0503 AVE 7688 (Aventis) 0504 M100240 (Aventis) Other Anti-Hypertension Drugs 0505 Z13752A (Zambon/GSK) 0533 MC4232 (University of Manitoba/Medicure) 0506 796406 (Zambon/GSK) Other Drugs from the PDR: Dual Neutral Endopeptidase and Endothelin Converting Indication=Hypertension Enzyme (NEP/ECE) Inhibitors 0507 SLV306 (Solvay) Accupril Tablets (Parke-Davis) 0534 Quinapril Hydrochloride NEP Inhibitors 05.08 Ecadotril (Bioproject) Accuretic Tablets (Parke-Davis) 0535 Hydrochlorothiazide, Quinapril Hydrochloride Aldosterone Antagonists 05.09 Eplerenone (Pharmacia) Aceon Tablets (2 mg, 4 mg., 8 mg) (Solvay) 0536 Perindopril Erbumine Renin Inhibitors 0510 Aliskiren (Novartis) Adalat CCTablets (Bayer) 0511 SPP 500 (Roche/Speedel) 0537 Nifedipine US 2010/0075894 A1 Mar. 25, 2010 24

Aldoclor Tablets (Merck) Coreg Tablets (GlaxoSmithKline) 0538 Chlorothiazide, Methyldopa 0557 Carvedilol Aldoril Tablets (Merck) Corzide 40/5 Tablets (King) 0539 Hydrochlorothiazide, Methyldopa 0558 Bendroflumethiazide, Nadolol Altace Capsules (King) Corzide 8015 Tablets (King) (0540 Ramipril 0559 Bendroflumethiazide, Nadolol Atacand Tablets (AstraZeneca LP) Covera-HS Tablets (Searle) 0541 Candesartan Cilexetil 0560 Verapamil Hydrochloride Atacand HCT 16-12.5 Tablets (AstraZeneca LP) Cozaar Tablets (Merck) 0542 Candesartan Cilexetil, Hydrochlorothiazide 0561 Losartan Potassium Atacand HCT 32-12.5 Tablets (AstraZeneca LP) Demadex Tablets and Injection (Roche Laboratories) 0543 Candesartan Cilexetil, Hydrochlorothiazide 0562 Torsemide Avalide Tablets (Bristol-Myers Squibb) Diovan HCT Tablets (Novartis) 0544 Hydrochlorothiazide, Irbesartan 0563 Hydrochlorothiazide, Valsartan Avapro Tablets (Bristol-Myers Squibb) Diovan Tablets (Novartis) 0545 Irbesartan 0564 Valsartan Avapro Tablets (Sanofi-Aventis) Diuril Oral Suspension (Merck) 0546 Irbesartan 0565 Chlorothiazide Benicar Tablets (Sankyo) Diuril Tablets (Merck) (0547 Olmesartan Medoxomil 0566 Chlorothiazide Benicar HCT Tablets (Sankyo) Dyazide Capsules (GlaxoSmithKline) 0548 Hydrochlorothiazide, Olmesartan Medoxomil 0567 Hydrochlorothiazide, Triamterene Blocadren Tablets (Merck) DynaCirc CR Tablets (Reliant) 0549. Timolol Maleate 0568 Isradipine Caduet Tablets (Pfizer) Furosemide Tablets (Mylan) 0550 Amlodipine Besylate, Atorvastatin Calcium 0569. Furosemide Captopril Tablets (Mylan) HydroDIURIL Tablets (Merck) 0551 Captopril (0570 Hydrochlorothiazide Cardene I.V. (ESP Pharma) Hytrin Capsules (Abbott) 0552. Nicardipine Hydrochloride (0571 Terazosin Hydrochloride Cardizem LA Extended Release Tablets (BioVail) Hyzaar 50-12.5 Tablets (Merck) 0553 Diltiazem Hydrochloride 0572 Hydrochlorothiazide, Losartan Potassium Catapres Tablets (Boehringer Ingelheim) Hyzaar 100-25 Tablets (Merck) 0554 Clonidine Hydrochloride 0573 Hydrochlorothiazide, Losartan Potassium Catapres-TTS (Boehringer Ingelheim) Indapamide Tablets (Mylan) 0555 Clonidine (0574 Indapamide Clorpres Tablets (Mylan Bertek) Inderal LA Long-Acting Capsules (Wyeth) 0556 Chlorthalidone, Clonidine Hydrochloride (0575 Propranolol Hydrochloride US 2010/0075894 A1 Mar. 25, 2010

InnoPran XL Capsules (Reliant) Norvasc Tablets (Pfizer) 0576 Propranolol Hydrochloride 0591 Amlodipine Besylate Inspira Tablets (Pfizer) Prinivil Tablets (Merck) 0577 Eplerenone 0592 Lisinopril Inversine Tablets (Targacept) Prinzide Tablets (Merck) 0578 Mecamylamine Hydrochloride 0593. Hydrochlorothiazide, Lisinopril Isoptin SR Tablets (Abbott) Sular Tablets (First Horizon) 0579. Verapamil Hydrochloride 0594 Nisoldipine Lotensin Tablets (Novartis) Tarka Tablets (Abbott) 0595 Trandolapril, Verapamil Hydrochloride 0580 Benazepril Hydrochloride TevetenTablets (BioVail) Lotensin HCT Tablets (Novartis) 0596 Eprosartan Mesylate 0581 Benazepril Hydrochloride, Hydrochlorothiazide Teveten HCT Tablets (BioVail) Lotrel Capsules (Novartis) 0597 Eprosartan Mesylate, Hydrochlorothiazide 0582 Amlodipine Besylate, Benazepril Hydrochloride Tiazac Capsules (Forest) Mavik Tablets (Abbott) 0598. Diltiazem Hydrochloride 0583. Trandolapril Timolide Tablets (Merck) Maxzide Tablets (Mylan Bertek) 0599 Hydrochlorothiazide, Timolol Maleate 0584) Hydrochlorothiazide, Triamterene Toprol-XL Tablets (AstraZeneca LP) Maxzide-25 mg Tablets (Mylan Bertek) 0600 Metoprolol Succinate 0585) Hydrochlorothiazide, Triamterene Uniretic Tablets (Schwarz) Micardis Tablets (Boehringer Ingelheim) 0601 Hydrochlorothiazide, Moexipril Hydrochloride 0586 Telmisartan Univasc Tablets (Schwarz) 0602 Moexipril Hydrochloride Micardis HCT Tablets (Boehringer Ingelheim) 0587 Hydrochlorothiazide, Telmisartan Vaseretic Tablets (Merck) 0603 Enalapril Maleate, Hydrochlorothiazide Midamor Tablets (Merck) Vasotec I.V. Injection (Merck) 0588 Amiloride Hydrochloride 0604) Enalapril Maleate Moduretic Tablets (Merck) Verelan PM Capsules (Schwarz) 0589 Amiloride Hydrochloride, Hydrochlorothiazide 0605 Verapamil Hydrochloride Nadolol Tablets (Mylan) Zaroxolyn Tablets (Celltech) 0590 Nadolol 06.06 Metolazone

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 2

<21 Os SEQ ID NO 1 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: US 2010/0075894 A1 Mar. 25, 2010 26

- Continued <223> OTHER INFORMATION: synthetic primer <4 OOs, SEQUENCE: 1 tggagttcCC Cagattgaag

<210s, SEQ ID NO 2 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic primer <4 OOs, SEQUENCE: 2 cctgacccac ctittittctica

1. A method of treating or preventing a condition selected 20. The method of claim 1, wherein the agent is a derivative from the group consisting of obesity, insulin resistance, or salt of trimethylamine N-oxide (TMAO). hyperglycemia, and type 2 diabetes, the method comprising: 21. The method of claim 1, wherein the agent is of the administering to an animal an agent known to reduce ER formula: StreSS. 2. The method of claim 1 further comprising administering an agent selected from the group consisting of anti-diabetic agents, anti-obesity agents, anti-dyslipidemia agents, anti atherosclerosis agents, and anti-hypertensive agents. 3. The method of claim 2, wherein the anti-diabetic agentis selected from the group consisting of biguanides, metformin, Sulfonylureas, insulin, analogs of insulin, PPARg agonists, meglitinides, and DPP-IV inhibitors. wherein 4. The method of claim 2, wherein the anti-obesity agent is R. R. and R are independently hydrogen, halogen, or selected from the group consisting of pancreatic lipase inhibi lower C-C alkyl, or a pharmaceutically-acceptable tors, serotonin reuptake inhibitors, norepinephrine reuptake salt thereof, or a mixture thereof. inhibitors, noradrenergic anorectic agents, peripherally act 22. (canceled) ing agents, centrally acting agents, and thermogenic agents. 23. The method of claim 1, wherein the agent is phenyl 5. The method of claim 2, wherein the anti-dyslipidemia butyric acid (PBA) or a derivative, salt, or isomer of PBA. agent or anti-atherosclerosis agent is selected from the group 24. (canceled) consisting of HMG-CoA reductase inhibitors, niacin, anti 25. The method of claim 1, wherein the agent is of the platelets, ACE inhibitors, aspirin, analogs of aspirin, and formula: MCP-1 inhibitors. 6. The method of claim 2, wherein the anti-hypertensive agent is selected from the group consisting of diuretics, beta R R3 O blockers, Ca' channel blockers, ACE inhibitors, and AT-II inhibitors. R- OH 7. (canceled) Rio R4 8. (canceled) 9. (canceled) 10. The method of claim 1, wherein the animal is a mam wherein n is 1 or 2; mal. Ro is aryl, heteroaryl, or phenoxy, the aryl and phenoxy 11. The method of claim 1, wherein the animal is a human. being unsubstituted or substituted with, independently, 12. The method of claim 1, wherein the step of administer one or more halogen, hydroxy or lower alkyl; ing comprises administering an agent orally, administering an RandR are independently H. lower alkoxy, hydroxy, agent parenterally or administering an agent intravenously. lower alkyl or halogen; and 13. (canceled) R and Rare independently H. lower alkyl, lower alkoxy 14. (canceled) or halogen; or a pharmaceutically-acceptable derivative 15. The method of claim 1, wherein the agent is a chemical or salt thereof. chaperone selected from the group consisting of glycerol, 26-33. (canceled) D.O, dimethylsulfoxide (DMSO), glycine betaine (betaine), 34. The method of claim 1, wherein the agent is taurour glycerolphosphocholine (GPC), methylamines, and trim sodeoxycholic acid (TUDCA) or a derivative, salt, or isomer ethylamine N-oxide (TMAO. of TUDCA. 16-19. (canceled) 35. (canceled) US 2010/0075894 A1 Mar. 25, 2010 27

36. The method of claim 1, wherein the agent is of the 54. (canceled) formula: 55. A method of screening for agents that prevent ER stress, the method comprising steps of: providing an agent to be screened; contacting the agent with a cell; CH3 Subsequently contacting the cell contacted with the agent O with an ER stress inducer; and determining whether ER stress markers are reduced. CH3 56. The method of claim 55, wherein the ER stress inducer RN is selected from the group consisting of tunicamycin and )-R, thapSigargin. HO OH R 57. (canceled) 58. (canceled) 59. A pharmaceutical composition comprising (1) an agent wherein R is —H or C-C alkyl: known to reduce ER stress, and (2) an agent selected from the R is —CH2—SOR and R is —H; or R is —COOH group consisting of anti-diabetic agents, anti-obesity agents, and R is —CH, CH, CONH, antidyslipidemia agents, anti-atherosclerosis agents, and CH CONH2, —CH2—CH2—SCH or —CH2— anti-hypertensive agents. S CH-COOH; and 60-64. (canceled) R3 is —H or the residue of a basic amino acid, or 65. A method of reducing ER stress or treating or prevent a pharmaceutically acceptable salt or derivative thereof. ing a disease associated with ER stress, the method compris 37. (canceled) ing steps of 38. (canceled) 39. The method of claim 1, wherein the agent is adminis administering to an animal an agent selected from the tered at a dose ranging from 100 mg/kg/day to 5 g/kg/day, a group consisting of PBA, TUDCA, and derivatives dose ranging from 500 mg/kg/day to 3 g/kg/day or a dose thereof. ranging from 500 mg/kg/day to 1 g/kg/day. 66. A method of diagnosing insulin resistance, hypergly 40. (canceled) cemia, or type 2 diabetes, the method comprising: 41. (canceled) measuring the level of expression of at least one ER stress 42. A method of treating or preventing obesity, the method marker, wherein an increase in the level of the ER stress comprising administering to a human an agent known to marker indicates that the Subject is at risk of insulin reduce ER stress. resistance, hyperglycemia, or type 2 diabetes. 43. A method of reducing blood glucose levels; or increas 67. The method of claim 66, wherein the ER stress marker ing insulin sensitivity, the method comprising administering is selected from the group consisting of spliced forms of to an animal an agent selected from the group consisting of XBP-1, phosphorylation status of PERK, phosphorylation of PBA, TUDCA, and derivatives thereof. eIF2a, mRNA levels of GRP78/BIP protein levels of GRP78/ 44. (canceled) BIP, and JNK activity. 45. A method of Screening for agents to: treat or prevent 68. (canceled) obesity, insulin resistance, or diabetes; modulate insulin 69. A method of modulating PERK, IRE-1a. JNK, IRS-1, action or insulin receptor signaling; or reduce ER stress, the IRS-2, Akt, or insulin receptor activity comprising: method comprising steps of administering to an animal an agent selected from the providing an agent to be screened; group consisting of PBA, TUDCA, and derivatives contacting the agent with a cell; and thereof determining whether ER stress markers are reduced. 70-76. (canceled) 46. (canceled) 77. A method of increasing insulin action or insulin recep 47. The method of claim 45, wherein ER stress markers are tor signaling, the method comprising: selected from the group consisting of spliced forms of XBP-1, administering to an animal an agent selected from the phosphorylation status of PERK, phosphorylation of eIF2a, group consisting of PBA, TUDCA, and derivatives mRNA levels of GRP78/BIP protein levels of GRP78/BIP. thereof. and JNK activity. 78. (canceled) 48. The method of claim 45, wherein the cell is a mamma 79. A method of modulating insulin receptor signaling lian cell, a human cell, an adipocyte or a hepatocyte. comprising: 49-51. (canceled) administering to an animal an agent known to reduce ER 52. The method of claim 45, wherein the cell is experienc StreSS. ing ER stress or has been treated with tunicamycin or thapsi 80-82. (canceled) gargin to induce ER stress. 53. (canceled)